Patent Application
20220220536
The instant application contains a sequence listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on 24 Mar. 2022, is named sequences_rna_finalmwk1a_ST25.txt and is 791,000 bytes in size.
The present disclosure relates to nucleic acid compositions and methods of use thereof for detecting RNA such as viral RNA and fragments thereof. More specifically, the present disclosure relates to methods of altering a nucleic acid shape or conformation such as a DNA nanoswitch with one or more ribonucleic acids and/or detecting or identifying the one or more ribonucleic acids.
Newly emerging or re-emerging viruses pose significant challenges to health care systems, particularly as globalization has contributed to the rampant spread of these viruses (See e.g., K. F. Smith, et al., Globalization of human infectious disease. Ecology. 88, 1903-1910 (2007)). RNA viruses are frequently the cause of sweeping outbreaks as these viruses have high mutation rates and thus evolve rapidly (See e.g., R. Sanjuán, et al., Viral Mutation Rates. Journal of Virology. 84, 9733-9748 (2010); and D. A. Steinhauer, J. J. Holland, Rapid Evolution of RNA Viruses. Annual Review of Microbiology. 41, 409-431 (1987)). Examples of this include the annual influenza outbreak, Ebola virus, Zika virus (ZIKV) and the SARS-CoV-2 virus responsible for the COVID-19 pandemic. Technological advancements in structural biology and genomics have been important for identifying viruses, and for advancing fundamental viral research and antiviral therapeutics (See e.g., H. D. Marston, et al., Emerging Viral Diseases: Confronting Threats with New Technologies. Science Translational Medicine. 6, 253ps10-253ps10 (2014)). However, the inventors have observed that clinical methods for robust, low-cost and rapid detection of viral infections remain a major challenge for emergent viruses, especially in resource limited areas.
Detection of RNA viruses in the clinical setting is typically performed using either immunological detection based on enzyme-linked immunosorbent assay (ELISA) to detect IgM antibodies or nucleic acid testing (NAT) based on a reverse transcription polymerase chain reaction (RT-PCR) assay to detect viral RNA. (See e.g., K. R. Jerome, Lennette's Laboratory Diagnosis of Viral Infections (CRC Press, 2016); V. M. Corman, et al., Assay optimization for molecular detection of Zika virus. Bull World Health Organ. 94, 880-892 (2016); and D. Musso, D. J. Gubler, Zika Virus. Clinical Microbiology Reviews. 29, 487-524)(2016); D. J. Clark, et al., The current landscape of nucleic acid tests for filovirus detection. Journal of Clinical Virology. 103, 27-36 (2018)). Diagnosing RNA viruses, is made challenging by several factors including a limited time window for detection, low or varying viral load, cross-reactive IgM antibodies, and laboratory resources. The detection time windows can vary widely from as short as a few days to as long as several months (See e.g., K. R. Jerome, Lennette's Laboratory Diagnosis of Viral Infections (CRC Press, 2016)), and molecular detection techniques are usually most reliable if performed within the first two weeks of the disease (See e.g., K. James, Immunoserology of infectious diseases. Clinical Microbiology Reviews. 3, 132-152 (1990); and L. R. Petersen, D. J. Jamieson, A. M. Powers, M. A. Honein, Zika Virus. New England Journal of Medicine. 374, 1552-1563 (2016)). Depending on the timing of testing relative to infection, even highly sensitive NAT assays may still produce false negative or false positive results (See e.g., V. M. Corman, et al., Assay optimization for molecular detection of Zika virus. Bull World Health Organ. 94, 880-892 (2016)). On the other hand, results from IgM serology tests often cannot distinguish related viruses or different strains of the same virus due to cross-reactivity of IgM antibodies, thus leading to false positive results (See W. Dejnirattisai, et al., Dengue virus sero-cross-reactivity drives antibody-dependent enhancement of infection with zika virus. Nature Immunology. 17, 1102-1108 (2016); and C. R. Woods, False-Positive Results for Immunoglobulin M Serologic Results: Explanations and Examples. J Pediatric Infect Dis Soc. 2, 87-90 (2013)). These detection challenges are further exacerbated when outbreaks occur in low resource settings where infrastructure for these lab-intensive tests can be lacking, accelerating the spread of disease (See e.g., D. Musso, D. J. Gubler, Zika Virus. Clinical Microbiology Reviews. 29, 487-524 (2016), and J. V. Lazarus, et al., Too many people with viral hepatitis are diagnosed late—with dire consequences. Nat Rev Gastroenterol Hepatol. 16, 451-452 (2019)). In response to some of these challenges, new techniques are being developed to detect emerging viruses. Among these are methods that adopt nanoparticles (M. S. Draz, H. Shafiee, Applications of gold nanoparticles in virus detection. Theranostics. 8, 1985-2017 (2018)), graphene-based biosensors (See e.g., S. Afsahi, et al., Novel graphene-based biosensor for early detection of Zika virus infection. Biosensors and Bioelectronics. 100, 85-88 (2018)), and CRISPR-based methods (See J. S. Gootenberg, et al., Nucleic acid detection with CRISPR-Cas13a/C2c2. Science, eaam9321 (2017); and K. Pardee, et al., Rapid, Low-Cost Detection of Zika Virus Using Programmable Biomolecular Components. Cell. 165, 1255-1266 (2016)), to name a few. Many of these proposed strategies, although based on cutting-edge technology, require multiple reactions or signal transformation steps.
DNA nanoswitches are versatile nucleic acid complexes typically including a nucleic acid, either single- or double-stranded, which is modified to contain preselected segments of nucleic acids and designed to assume a linear (or open) conformation or a looped (or closed) formation depending upon the presence of an oligonucleotide such as an oligonucleotide-of-interest. DNA nanoswitches have been described in U.S. Patent Publication No. 2018/0223344 (herein entirely incorporated by reference) however it has not been heretofore contemplated to alter the conformation of a DNA nanoswitch as described herein such that when the DNA nanoswitch is combined or contacted with a ribonucleic nucleic acid-of-interest, it forms a DNA nanoswitch-ribonucleic acid complex suitable for providing a signal for RNA viral detection/identification as described herein, such as when contacted with viral RNA or one or more portions thereof.
There is a continuing need for robust methods of detecting RNA and identifying viral RNA.
The present disclosure relates to a method of reconfiguring or changing the shape of a nucleic acid in accordance with the present disclosure, which is useful for, inter alia, detecting ribonucleic acid-of-interest such as viral ribonucleic acids or portions thereof. In embodiments, the present disclosure is directed towards a method of detecting an RNA virus by reconfiguring or changing the shape of a nucleic acid including: contacting a deoxyribonucleic acid (DNA) nanoswitch having a first conformation characterized as open with a biological specimen to form a mixture, wherein when the mixture includes a ribonucleic acid-of-interest, the first conformation changes to a second conformation characterized as closed; processing the mixture under conditions sufficient to separate the first conformation, and when present, the second conformation; and reacting the first conformation, and when present, the second conformation with an indicator under conditions sufficient to form a signal.
In some embodiments, the present disclosure relates to a method of reconfiguring a nucleic acid including: contacting a deoxyribonucleic acid (DNA) nanoswitch having a first conformation, with a ribonucleic acid to form a DNA nanoswitch-nucleic acid complex having a second conformation, wherein the second conformation is characterized as locked; processing a mixture under conditions sufficient to separate the first conformation and the second conformation; and contacting the first conformation and second conformation with an indicator under conditions sufficient to form a signal. In some embodiments, the signal is predetermined to show a presence or absence of ribonucleic acid-of-interest.
In some embodiments, the present disclosure relates to a nucleic acid, including: a DNA nanoswitch-nucleic acid complex including a deoxyribonucleic acid (DNA) nanoswitch and one or more ribonucleic acid binding sites, wherein the DNA nanoswitch has a first conformation characterized as open, and a second conformation characterized as closed when in a presence of one or more ribonucleic acids-of-interest.
In embodiments, the present disclosure includes a DNA nanoswitch suitable for forming a DNA nanoswitch-ribonucleic acid complex, including: a scaffold including a plurality of nucleotides or a polynucleotide sequence; a plurality of backbone oligonucleotides hybridized to the scaffold to form a backbone polynucleotide; a first detector strand including a nucleic acid sequence having a first segment hybridized to the scaffold or the backbone polynucleotide, and a second segment characterized as an overhang, wherein the second segment is hybridizable to, when present a first segment of a preselected target RNA nucleotide sequence or target RNA polynucleotide-of-interest; and a second detector strand comprising a nucleic acid sequence having a first segment hybridized to the scaffold or the backbone polynucleotide, and a second segment characterized as an overhang, wherein the second segment is hybridizable to, when present, a second segment of the preselected target RNA nucleotide sequence or the target RNA polynucleotide-of-interest.
In embodiments, the present disclosure includes a kit, including: one or more DNA nanoswitches of the present disclosure, wherein, when present, the DNA nanoswitch hybridizes a viral RNA or a fragment thereof; and a separation medium, and optionally a buffer solution.
The illustrative aspects of the present disclosure are designed to solve the problems herein described and/or other problems not discussed.
The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.
Embodiments of the present disclosure, briefly summarized above and discussed in greater detail below, can be understood by reference to the illustrative embodiments of the disclosure depicted in the appended drawings. However, the appended drawings illustrate only typical embodiments of the disclosure and are therefore not to be considered limiting of scope, for the disclosure may admit to other equally effective embodiments.
The present disclosure is directed towards compositions, kits, and methods of detecting a virus by reconfiguring a nucleic acid such as a DNA nanoswitch including: contacting a deoxyribonucleic acid (DNA) nanoswitch having a first conformation characterized as open with a biological specimen to form a mixture, wherein when the mixture includes a ribonucleic acid-of-interest, the first conformation changes to a second conformation characterized as closed; processing the mixture under conditions sufficient to separate the first conformation, and when present, the second conformation; and reacting the first conformation, and when present, the second conformation with an indicator under conditions sufficient to form a signal. Advantages of the various embodiments of the present disclosure include overcoming biosensing challenges by using programmable DNA nanoswitches for detection of viral RNA at clinically relevant levels.
Embodiments of the compositions, kits, and methods of the present disclosure are validated in the examples below where, e.g., a viral RNA detection strategy was performed using ZIKV as a model RNA virus. Zika has high global health relevance and is a continued threat due to its re-emerging mosquito-borne nature. Although ZIKV infections are typically associated with mild symptoms, they have been linked to devastating birth defects associated with intrauterine infections, development of Guillian-Barré syndrome in adults, and the possibility of sexual transmission (see e.g., D. Musso, D. J. Gubler, Zika Virus. Clinical Microbiology Reviews. 29, 487-524 (2016); L. R. Petersen, et al., Zika Virus. New England Journal of Medicine. 374, 1552-1563 (2016)). Moreover, despite significant advances in understanding the molecular biology of ZIKV, there is still a lack of antiviral drugs and vaccines, making robust detection of ZIKV vital to controlling the spread of the disease and implementing early treatments (See e.g., A. D. T. Barrett, Current status of Zika vaccine development: Zika vaccines advance into clinical evaluation. npj Vaccines. 3, 24 (2018)). Non-limiting example of ribonucleic acid-of-interest for identification or detection herein include RNA from one or more riboviruses, or RNA from one or more RNA viruses. Non-limiting examples of RNA viruses include: virus that causes the common cold, influenza virus, SARS virus, SARS-CoV-2, Dengue virus, hepatitis C virus, hepatitis E virus, West Nile fever virus, Ebola virus, rabies virus, polio virus, measles virus, Zika virus, as well as variants and strains of these viruses. In some embodiments, RNA viruses include those in which The International Committee on Taxonomy of Viruses (ICTV) classifies as RNA viruses such as those that belong to Group III, Group IV or Group V of the Baltimore classification system of classifying viruses and does not consider viruses with DNA intermediates in their life cycle as RNA viruses. In embodiments, viruses with RNA as their genetic material which also include DNA intermediates in their replication cycle, Retrovirus, and include Group VI of the Baltimore classification such as HIV-1 and HIV-2 may also be identified in accordance with the methods of the present disclosure. In some embodiments; the ribonucleic acid of interest may include ribonucleic acid from double stranded RNA viruses, single stranded plus sense RNA viruses or single stranded negative sense RNA viruses. In embodiments, Zika virus is an example of a single-stranded RNA virus of the Flaviviridae family, genus Flavivirus suitable for identification and or detection in accordance with the present disclosure. The complete genome of the Zika virus is known. (See e.g., NCIB accession no. NC_012532).
In embodiments, positive-strand RNA virus, such as sense-strand RNA virus is suitable for detection and/or identification in accordance with the present disclosure. Examples of positive strand RNA viruses include polio virus, Coxsackie virus, echovirus, and the like. In embodiments, negative-strand RNA virus, such as antisense-strand RNA virus (e.g., -ssRNA viruses) is suitable for detection and/or identification in accordance with the present disclosure. Non-limiting examples of ssRNA viruses include Ebola virus, hantaviruses, influenza viruses, Lassa fever virus, rabies virus, family members of these, variants of these, and combinations thereof.
In embodiments, the compositions and methods of the present disclosure are suitable for detecting the presence of viral RNA based on using DNA nanoswitches designed to undergo a conformational change (from linear to looped) upon binding a target viral RNA. In embodiments, the presence of the viral RNA would be indicated by shifted migration of the looped nanoswitch by gel electrophoresis. In some embodiments, the methods include a common nucleic acid staining of the nanoswitch itself that can intercalate thousands of dye molecules to provide an inherently strong signal. In embodiments, as applied here to viral RNA detection, challenges of detecting a long viral RNA (>10,000 nucleotides) in clinically relevant samples have been overcome.
Embodiments of the present disclosure include an RNA fragmentation process, wherein e.g., large RNA strands are separated into small RNA segments, a signal multiplication feature, use of an algorithm for choosing target sequences, and workflows for measuring viral loads in biological and mock clinical samples with or without RNA pre-amplification. In some embodiments, multiplexing is used to detect multiple viruses simultaneously from a single sample and demonstrate high specificity even between closely related strains of virus, such as Zika. In some embodiments, DNA nanoswitches (
In embodiments, the compositions and methods of the present disclosure herein relate to altering the conformation of one or more DNA nanoswitches for signaling depending upon signal needs. In some embodiments, the present disclosure relates to a method of detecting a virus by reconfiguring a nucleic acid including: contacting a deoxyribonucleic acid (DNA) nanoswitch having a first conformation characterized as open with a biological specimen to form a mixture, wherein when the mixture includes a ribonucleic acid-of-interest, the first conformation changes to a second conformation characterized as closed. In embodiments, a process flow subsequently includes processing the mixture under conditions sufficient to separate the first conformation, and when present, the second conformation; and reacting the first conformation, and when present, the second conformation with an indicator under conditions sufficient to form a signal. In embodiments, reacting may be as simple as adding a dye to make a first conformation and/or second confirmation detectable. In embodiments, the ribonucleic acid-of-interest is derived from an RNA virus in a biological specimen or sample.
The embodiments of the present disclosure may advantageously provide nucleic acid complexes configured to provide one or more useful signals such as the presence or absence of viral RNA. In embodiments, the present disclosure advantageously provides improved methods, compositions, and assays for the detection, or identification of one or more RNA targets-of-interest such as one or more viral RNAs, or species or fragments thereof, and combinations thereof.
As used in the present specification, the following words and phrases are generally intended to have the meanings as set forth below, except to the extent that the context in which they are used indicates otherwise.
As used herein, the singular forms “a”, “an”, and “the” include plural references unless the context clearly dictates otherwise. Thus, for example, references to “a compound” include the use of one or more compound(s). “A step” of a method means at least one step, and it could be one, two, three, four, five or even more method steps.
As used herein the terms “about,” “approximately,” and the like, when used in connection with a numerical variable, generally refers to the value of the variable and to all values of the variable that are within the experimental error (e.g., within the 95% confidence interval [CI 95%] for the mean) or within ±10% of the indicated value, whichever is greater.
As used herein, the terms “bind” and “binding” generally refer to a non-covalent interaction between a pair of partner molecules or portions thereof that exhibit mutual affinity or binding capacity. In embodiments, binding can occur such that the partners are able to interact with each other to a substantially higher degree than with other, similar substances. This specificity can result in stable complexes that remain bound during handling steps such as chromatography, centrifugation, filtration, and other techniques typically used for separations and other processes.
The terms “deoxyribonucleotide” and “DNA” refer to a nucleotide or polynucleotide including at least one ribosyl moiety that has an H at the 2′ position of a ribosyl moiety. In embodiments, a deoxyribonucleotide is a nucleotide having an H at its 2′ position.
By “hybridizable” or “complementary” or “substantially complementary” a nucleic acid (e.g. RNA, DNA) includes a sequence of nucleotides that enables it to non-covalently bind, i.e. form Watson-Crick base pairs and/or G/U base pairs, “anneal”, or “hybridize,” to another nucleic acid in a sequence-specific, antiparallel, manner (e.g., a nucleic acid specifically binds to a complementary nucleic acid) under the appropriate in vitro and/or in vivo conditions of temperature and solution ionic strength. Standard Watson-Crick base-pairing includes: adenine/adenosine) (A) pairing with thymidine/thymidine (T), A pairing with uracil/uridine (U), and guanine/guanosine) (G) pairing with cytosine/cytidine (C). In addition, for hybridization between two RNA molecules, and for hybridization of a DNA molecule with an RNA molecule (e.g., when a DNA nanoswitch base pairs with a target RNA, etc.): G can also base pair with U. For example, G/U base-pairing is partially responsible for the degeneracy (i.e., redundancy) of the genetic code in the context of tRNA anti-codon base-pairing with codons in mRNA. In embodiments, hybridization requires that the two nucleic acids contain complementary sequences, although mismatches between bases are possible. The conditions appropriate for hybridization between two nucleic acids depend on the length of the nucleic acids and the degree of complementarity, variables well known in the art. The greater the degree of complementarity between two nucleotide sequences, the greater the value of the melting temperature (Tm) for hybrids of nucleic acids having those sequences. Typically, the length for a hybridizable nucleic acid is 8 nucleotides or more (e.g., 10 nucleotides or more, 12 nucleotides or more, 15 nucleotides or more, 20 nucleotides or more, 22 nucleotides or more, 25 nucleotides or more, or 30 nucleotides or more). It is understood that the sequence of a polynucleotide need not be 100% complementary to that of its target nucleic acid to be specifically hybridizable. Moreover, a polynucleotide may hybridize over one or more segments such that intervening or adjacent segments are not involved in the hybridization event (e.g., a loop structure or hairpin structure, a ‘bulge’, and the like). A polynucleotide can include 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, 99% or more, 99.5% or more, or 100% sequence complementarity to a target region within the target nucleic acid sequence to which it will hybridize. For example, an antisense nucleic acid in which 18 of 20 nucleotides of the antisense compound are complementary to a target region, and would therefore specifically hybridize, would represent 90 percent complementarity. The remaining noncomplementary nucleotides may be clustered or interspersed with complementary nucleotides and need not be contiguous to each other or to complementary nucleotides. Percent complementarity between particular stretches of nucleic acid sequences within nucleic acids can be determined using any convenient method. Example methods include BLAST programs (basic local alignment search tools) and PowerBLAST programs (Altschul et al., J. Mol. Biol., 1990, 215, 403-410; Zhang and Madden, Genome Res., 1997, 7, 649-656) or by using the Gap program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, Madison Wis.), e.g., using default settings, which uses the algorithm of Smith and Waterman (Adv. Appl. Math., 1981, 2, 482-489).
The terms “elute” and “eluting” refer to the disruption of non-covalent interactions between partner molecules such that the partners become unbound from one another. In embodiments, the disruption can be effected via introduction of a competitive binding species, introduction of RNase, or via a change in environmental conditions (e.g., ionic strength, pH, or other conditions).
As used herein, the term “forming a mixture” refers to the process of bringing into contact at least two distinct species such that they mix together and interact. “Forming a reaction mixture” and “contacting” refer to the process of bringing into contact at least two distinct species such that they mix together and can react, either modifying one of the initial reactants or forming a third, distinct, species, a product. It should be appreciated, however, the resulting reaction product can be produced directly from a reaction between the added reagents or from an intermediate from one or more of the added reagents which can be produced in the reaction mixture. “Conversion” and “converting” refer to a process including one or more steps wherein a species is transformed into a distinct product.
An “isolated nucleic acid molecule” is a polymer of RNA or DNA that is single- or double-stranded, optionally containing synthetic, non-natural or altered nucleotide bases. An isolated nucleic acid molecule in the form of a polymer of DNA may be one or more segments of cDNA, genomic DNA or synthetic DNA.
The term “nucleotide” refers to a ribonucleotide or a deoxyribonucleotide or modified form thereof, as well as an analog thereof.
The term “nanoswitch” refers to a nucleic acid molecule, either single- or double-stranded, which is modified to contain segments of nucleic acids in a manner that would not otherwise exist in nature, that assumes a linear (or open) conformation in the absence of a predetermined nucleic acid or a looped (or closed) formation in the presence of the same predetermined nucleic acid.
Nucleic acid construct: The term “nucleic acid construct” as used herein refers to a nucleic acid molecule, either single- or double-stranded, which is modified to contain segments of nucleic acids in a manner that would not otherwise exist in nature.
As used herein, the term “nucleic acid molecule” refers to any molecule containing multiple nucleotides (e.g., molecules including a sugar (e.g., ribose or deoxyribose) linked to a phosphate group and to an exchangeable organic base, which is either a substituted pyrimidine (e.g., cytosine (C), thymine (T) or uracil (U)) or a substituted purine (e.g., adenine (A) or guanine (G)). As described further below, bases include C, T, U, C, and G, as well as variants thereof. As used herein, the term refers to ribonucleotides (including oligoribonucleotides (ORN)) as well as deoxyribonucleotides (including oligodeoxynucleotides (ODN)). The term shall also include polynucleosides (e.g., a polynucleotide minus the phosphate) and any other organic base containing polymer. Nucleic acid molecules can be obtained from existing nucleic acid sources (e.g., genomic or cDNA), but include synthetic (e.g., produced by oligonucleotide synthesis). In embodiments, the terms “nucleic acid” “nucleic acid molecule” and “polynucleotide” may be used interchangeably herein, and refer to both RNA and DNA, including cDNA, genomic DNA, synthetic DNA, and DNA (or RNA) containing nucleic acid analogs. Polynucleotides can have any three-dimensional structure. A nucleic acid can be double-stranded or single-stranded (i.e., a sense strand or an antisense strand). Non-limiting examples of polynucleotides include genes, gene fragments, exons, introns, messenger RNA (mRNA) and portions thereof, transfer RNA, ribosomal RNA, siRNA, micro-RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers, as well as nucleic acid analogs.
In embodiments, the term “oligonucleotide” refers to a polynucleotide of between 4 and 100 nucleotides of single- or double-stranded nucleic acid (e.g., DNA, RNA, or a modified nucleic acid). However, for the purposes of this disclosure, there is no upper limit to the length of an oligonucleotide. Oligonucleotides are also known as “oligomers” or “oligos” and can be isolated from genes, transcribed (in vitro and/or in vivo), or chemically synthesized.
The term “isolated” means a substance in a form or environment that does not occur in nature. Non-limiting examples of isolated substances include (1) any non-naturally occurring substance, (2) any substance such as a variant, nucleic acid, protein, peptide or cofactor, that is at least partially removed from one or more or all of the naturally occurring constituents with which it is associated in nature; (3) any substance modified by the hand of man relative to that substance found in nature; or (4) any substance modified by increasing the amount of the substance relative to other components with which it is naturally associated.
The term “polynucleotide” refers to polymers of nucleotides. In embodiments, the term polynucleotide includes but is not limited to DNA, RNA, DNA/RNA hybrids including polynucleotide chains of regularly and irregularly alternating deoxyribosyl moieties and ribosyl moieties (e.g., wherein alternate nucleotide units have an —OH, then and —H, then an —OH, then an —H, and so on at the 2′ position of a sugar moiety), and modifications of these kinds of polynucleotides wherein the attachment of various entities or moieties to the nucleotide units at any position are included.
The term “polyribonucleotide” refers to a polynucleotide including two or more modified or unmodified ribonucleotides and/or their analogs.
The term “ribonucleotide” and the phrase “ribonucleic acid” (RNA), refer to a modified or unmodified nucleotide or polynucleotide including at least one ribonucleotide unit. A ribonucleotide unit includes an oxygen attached to the 2′ position of a ribosyl moiety having a nitrogenous base attached in N-glycosidic linkage at the 1′ position of a ribosyl moiety, and a moiety that either allows for linkage to another nucleotide or precludes linkage.
The terms “sequence identity”, “identity” and the like as used herein with respect to polynucleotide or polypeptide sequences refer to the nucleic acid residues or amino acid residues in two sequences that are the same when aligned for maximum correspondence over a specified comparison window. Thus, “percentage of sequence identity”, “percent identity” and the like refer to the value determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide or polypeptide sequence in the comparison window may include additions or deletions (e.g., gaps) as compared to the reference sequence (which does not include additions or deletions) for optimal alignment of the two sequences. The percentage may be calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the results by 100 to yield the percentage of sequence identity.
It would be understood that, when calculating sequence identity between a DNA sequence and an RNA sequence, T residues of the DNA sequence align with, and can be considered “identical” with, U residues of the RNA sequence. For purposes of determining “percent complementarity” of first and second polynucleotides, one can obtain this by determining (i) the percent identity between the first polynucleotide and the complement sequence of the second polynucleotide (or vice versa), for example, and/or (ii) the percentage of bases between the first and second polynucleotides that would create canonical Watson and Crick base pairs.
In embodiments, the degree of sequence identity between a query sequence and a reference sequence is determined by: 1) aligning the two sequences by any suitable alignment program using the default scoring matrix and default gap penalty; 2) identifying the number of exact matches, where an exact match is where the alignment program has identified an identical amino acid or nucleotide in the two aligned sequences on a given position in the alignment; and 3) dividing the number of exact matches with the length of the reference sequence. In one embodiment, the degree of sequence identity between a query sequence and a reference sequence is determined by: 1) aligning the two sequences by any suitable alignment program using the default scoring matrix and default gap penalty; 2) identifying the number of exact matches, where an exact match is where the alignment program has identified an identical amino acid; or nucleotide in the two aligned sequences on a given position in the alignment; and 3) dividing the number of exact matches with the length of the longest of the two sequences. In some embodiments, the degree of sequence identity refers to and may be calculated as described under “Degree of Identity” in U.S. Pat. No. 10,531,672 starting at Column 11, line 56. U.S. Pat. No. 10,531,672 is incorporated by reference in its entirety. In embodiments, an alignment program suitable for calculating percent identity performs a global alignment program, which optimizes the alignment over the full-length of the sequences. In embodiments, the global alignment program is based on the Needleman-Wunsch algorithm (Needleman, Saul B.; and Wunsch, Christian D. (1970), “A general method applicable to the search for similarities in the amino acid sequence of two proteins”, Journal of Molecular Biology 48 (3): 443-53). Examples of current programs performing global alignments using the Needleman-Wunsch algorithm are EMBOSS Needle and EMBOSS Stretcher programs, which are both available on the world wide web at www.ebi.ac.uk/Tools/psa/. In some embodiments a global alignment program uses the Needleman-Wunsch algorithm, and the sequence identity is calculated by identifying the number of exact matches identified by the program divided by the “alignment length”, where the alignment length is the length of the entire alignment including gaps and overhanging parts of the sequences. In embodiments, the mafft alignment program is suitable for use herein.
The term “recombinant” when used herein to characterize a nucleic acid sequence such as a plasmid, vector, construct, or complex refers to an artificial combination of two otherwise separated segments of sequence, e.g., by chemical synthesis and/or by manipulation of isolated segments of nucleic acids by genetic engineering techniques.
The term “substantially purified,” as used herein, refers to a component of interest that may be substantially or essentially free of other components which normally accompany or interact with the component of interest prior to purification. By way of example only, a component of interest may be “substantially purified” when the preparation of the component of interest contains less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 10%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, or less than about 1 (by dry weight) of contaminating components. Thus, a “substantially purified” component of interest may have a purity level of about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or greater.
General methods in molecular and cellular biochemistry can be found in such standard textbooks as Molecular Cloning: A Laboratory Manual, 3rd Ed. (Sambrook et al., Harbor Laboratory Press 2001); Short Protocols in Molecular Biology, 4th Ed. (Ausubel et al. eds., John Wiley & Sons 1999); Protein Methods (Bollag et al., John Wiley & Sons 1996); Nonviral Vectors for Gene Therapy (Wagner et al. eds., Academic Press 1999); Viral Vectors (Kaplift & Loewy eds., Academic Press 1995); Immunology Methods Manual (I. Lefkovits ed., Academic Press 1997); and Cell and Tissue Culture: Laboratory Procedures in Biotechnology (Doyle & Griffiths, John Wiley & Sons 1998), the disclosures of which are incorporated herein by reference.
Before embodiments are further described, it is to be understood that this disclosure is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.
Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited.
In embodiments, the present disclosure relates to kits, compositions, or one or more methods of detecting an RNA virus by reconfiguring a nucleic acid such as a polynucleotide including: contacting a deoxyribonucleic acid (DNA) nanoswitch having a first conformation characterized as open with a biological specimen to form a mixture, wherein when the mixture includes a ribonucleic acid-of-interest, the first conformation changes to a second conformation characterized as closed; processing the mixture under conditions sufficient to separate the first conformation, and when present, the second conformation; and reacting the first conformation, and when present, the second conformation with an indicator under conditions sufficient to form a signal.
In embodiments, one or more DNA nanoswitches are preformed or preselected to combine with a preselected ribonucleotide-of-interest such as a preselected RNA, preselected RNA oligonucleotide, preselected RNA polynucleotide, viral RNA, fragments thereof, and combinations thereof. In embodiments, a DNA nanoswitch-nucleic acid complex suitable for use herein is a nucleic acid complex for use in detecting targets such as RNA virus. Targets are detected based on their interactions with the DNA nanoswitch-nucleic acid complex and the conformational changes that are induced in the nanoswitches and/or DNA nanoswitch-nucleic acid complex as result of such interactions. In embodiments, the DNA nanoswitch-nucleic acid complexes are designed so that in the absence of the target they typically maintain a linear (or open) conformation or shape and assume a looped (or closed) conformation or shape in the presence of a target such as viral RNA. In embodiments, conformation(s) refer to one or more structures of a DNA nanoswitch, including but not limited to any of the spatial arrangement which the atoms in the nanoswitch molecule may adopt and freely convert between, such as by rotation about individual single bonds therein. In embodiments, a linear or substantially linear conformation and a looped or closed conformation are detectable and physically separable from each other using various techniques including but not limited to gel electrophoresis. In the context of gel electrophoresis, the open and closed conformations migrate to different extents through a gel, and they can emit signal (such as when stained with a dye) from the gel in order to, in some embodiments, further inform that the target has altered the conformation of the nanoswitch. In embodiments, linear conformations migrate more faster through a gel medium under the same electrophoretic conditions, in comparison to looped or closed conformations formed of the linear conformations.
In embodiments, nanoswitches and DNA nanoswitch-nucleic acid complexes of the present disclosure are designed to detect and/or identify one or more preselected viral RNA targets. For example, in embodiments, a nanoswitch of the present disclosure is configured to bind to a binding partner to alter the shape of the nanoswitch, e.g., viral RNA. In embodiments, a predetermined viral RNA or fragment thereof is a binding partner with a DNA nanoswitch of the present disclosure. In embodiments, the binding partner may be one or more preselected ribonucleic acids, e.g. viral RNA, or fragments thereof that bind(s) to the nanoswitch and lock(s) the nanoswitch into a closed conformation. In embodiments, the preselected nucleic acid is RNA based on sequence complementarity. In embodiments, and as shown in
Non-limiting examples of viral RNA targets, and fragments thereof include targets derived from the viral RNA disposed within e.g., the virus that causes the common cold, influenza virus, SARS virus, SARS-CoV-2, Dengue virus, hepatitis C virus, hepatitis E virus, West Nile fever virus, Ebola virus, rabies virus, polio virus, measles virus, as well as variants and strains of these viruses. In embodiments, the viral RNA targets include one or more segments of RNA having a first length, such as a length between 20 nucleotides to 1000 nucleotides, which may be fragmented into a shorter length. In embodiments, the target RNA has a length of 20-200 nucleotides, 20-100 nucleotides, 20-60 nucleotides, 20-50 nucleotides, 20-40 nucleotides, 20-30 nucleotides, and the like.
In embodiments, detection of one or more viral RNA targets-of-interest is important for a variety of applications including for example in the fields of medicine and forensics. In some embodiments, the present disclosure provides a programmable nucleic acid-based nanoswitch that undergoes a pre-defined conformational change upon contact with a target ribonucleic acid such as viral RNA, converting a nanoswitch from a linear “open” state to a looped “closed” state (or conformation) within a DNA-nanoswitch-nucleic acid complex.
In embodiments, the looped “locked” state relates to a DNA complex or conformation that includes a combination of the DNA nanoswitch and a preselected ribonucleic acid combined to form a DNA nanoswitch-nucleic acid complex having a second conformation, wherein the second conformation is characterized as locked.
In embodiments, a DNA nanoswitch and/or DNA nanoswitch ribonucleic acid complex can be detected using separation techniques such as standard gel electrophoresis, which are capable of physically separating the open and locked conformations from each other and from other components in a mixture, and in some instances also are capable of facilitating isolation of DNA nanoswitch nucleic acid complex having a first conformation and/or nanoswitch having a second conformation different than the first conformation. In embodiments, other separation medium suitable for use herein may include liquid chromatography medium such as those used HPLC columns, or other medium such as those used in capillary electrophoresis.
In embodiments, the present disclosure demonstrates successful detection of one or more viral RNAs. In embodiments, the detection method can be accomplished quickly, including as demonstrated herein within 15 minutes from sample mixture to readout. The approach is a low cost and technically accessible, and thus well-suited for point-of-use detection.
In addition, the compositions of the present disclosure may also be used to simultaneously detect more than one viral RNA targets-of-interest. For example, the nanoswitch nucleic acid complex may be designed to include one or more nucleic acids configured to form the close looped shaped, wherein the one on more nucleic acids have different lengths and/or discernable structure. Variation in loop size may facilitate varying reaction conditions among a plurality of targets-of-interest such as different types of viral RNAs and facilitate detection between various viral RNAs.
In embodiments, a nucleic acid such as a DNA nanoswitch and/or DNA nanoswitch-ribonucleic acid complex, as described herein includes a scaffold nucleic acid hybridized in a sequence specific manner to a plurality of oligonucleotides. The scaffold and the oligonucleotides may be referred to herein as being single-stranded. In embodiments, prior to hybridization to each other, both nucleic acid species are single-stranded. In embodiments, upon hybridization, such as when subjected to conditions suitable for hybridization, a double-stranded nucleic acid is formed. Typically, the oligonucleotides hybridize to the scaffold nucleic acid in a consecutive, non-overlapping, manner.
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In embodiments, the DNA nanoswitch are modular complexes to which can be attached one or more nucleic acids of interest such as one or more oligonucleotides, one or more binding pairs of interest, and the like. The terms attach, link and conjugate are used interchangeably throughout this disclosure unless otherwise stated. In embodiments, as shown in
In embodiments, the nanoswitches of the present disclosure provided herein are stable in complex fluids such as but not limited to serum-containing samples, including up to 30% FBS. In some embodiments, nanoswitches for use herein are configured to convert from unbound to bound forms in the presence of complex fluids (e.g., 30% FBS). Moreover, the nanoswitches are also stable for an extended period of time. Once synthesized, the nanoswitches may be dried and stored for days, weeks or months. Similarly, DNA nanoswitch-nucleic acid complexes of the present disclosure may be stable for an extended period of time. Once synthesized, the DNA nanoswitch-nucleic acid complexes may be dried and stored for days, weeks or months.
In some embodiments, the nanoswitches of the present disclosure and/or DNA nanoswitch-ribonucleic acid complexes of the present disclosure can be made using nucleic acid nanostructure techniques such as but not limited to DNA origami. (See e.g., Rothemund P. W. K. (2006) Nature 440: 297-302; Douglas S. M. et al. (2009) Nature 459: 414-8). In embodiments, the nanoswitches of the present disclosure may be formed as described in U.S. Patent Publication No. 2018/0223344 entitled Compositions and Methods for Analyte Detection Using Nanoswitches published on 9 Aug. 2018 to Chandrasekaren et al. (herein entirely incorporated by reference).
In embodiments, scaffold nucleic acid suitable for use herein may be of any length sufficient to allow association (i.e., binding) and dissociation (i.e., unbinding) of binding partners to occur and to be distinguished from other association and/or dissociation events using the read out methods provided herein, including gel electrophoresis.
In embodiments, the scaffold nucleic acid is at least 500 nucleotides in length, and it may be as long as 50,000 nucleotides in length (or it may be longer). The scaffold nucleic acid may therefore be 1000-20,000 nucleotides in length, 1000-15,000 nucleotides in length, 1000-10,000 in length, or any range therebetween. In some embodiments, the scaffold ranges in length from about 5,000-10,000 nucleotides, and may be about 7000-7500 nucleotides in length or about 7250 nucleotides in length.
In some embodiments, the scaffold nucleic acid may also be non-naturally occurring nucleic acids such as polymerase chain reaction (PCR)-generated nucleic acids, rolling circle amplification (RCA)-generated nucleic acids, etc. In some embodiments, the scaffold nucleic acid is rendered single-stranded either during or post synthesis. Methods for generating a single-stranded scaffold include asymmetric PCR. Alternatively, double-stranded nucleic acids may be subjected to strand separation techniques in order to obtain the single-stranded scaffold nucleic acids. The scaffold nucleic acid may comprise DNA, RNA, DNA analogs, RNA analogs, or a combination thereof, provided it is able to hybridize in a sequence-specific and non-overlapping manner to the oligonucleotides or backbone oligonucleotides of the present disclosure. In some instances, the scaffold nucleic acid is a DNA.
In embodiments, the scaffold nucleic acid is hybridized to a plurality of oligonucleotides, such as the backbone oligonucleotides depicted in
In embodiments, the number of oligonucleotides will depend in part on the application, the length of the scaffold, and the length of the oligonucleotides themselves. In embodiments, the oligonucleotides are designed to be of approximately equal length. In some embodiments, the oligonucleotides may be about 20-100 nucleotides in length. The oligonucleotides may be, without limitation, about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 90 or about 100 nucleotides in length. In some embodiments, the oligonucleotides may be about 40-80 nucleotides in length. In some embodiments, the oligonucleotides may be about 60 nucleotides in length.
The number of oligonucleotides in the plurality may be about 70, about 80, about 90, about 100, about 110, about 120, about 130, about 140, about 150, about 160, about 170, about 180, about 190, about 200, about 300, about 400, about 400, about 500, about 600, about 700, about 800, about 900, or about 1000, without limitation.
In some embodiments, the nucleic acid complex may include the M13 ssDNA as the scaffold and about 120 oligonucleotides each equal to or about 60 nucleotides in length.
In embodiments, the oligonucleotides may be characterized as modified or unmodified or variable oligonucleotides. In embodiments, the variable oligonucleotides may be conjugated to reactive groups that are not normally present in a nucleic acid sequence, such as for example click chemistry reactive groups, or they may be conjugated to target-specific binding partners such as antibodies or antibody fragments, or they may include other moieties which are not typically present in an unmodified oligonucleotide. An example is a variable oligonucleotide including a phosphate at their 5′ end (referred to herein as a 5′ phosphate). Oligonucleotides having this latter modification are used herein in the detection of target nucleic acids, and in this context such oligonucleotides are referred to as “detector” strands since they are preselected to bind to nucleic acids of the interest via hybridization to form a DNA Nanoswitch-nucleic acid complex having a first conformation, wherein the first conformation is characterized as locked.
In some embodiments, the first and last oligonucleotides as well as “internal” oligonucleotides, typically at pre-defined positions along the length of the scaffold, may be modified oligonucleotides. The position of the variable oligonucleotides may be, but are not necessarily, evenly distributed along the length of the scaffold
Binding interactions and looped conformations are shown in
In embodiments, a nanoswitch may include a first and a second oligonucleotide such as a first and second detector strand that together hybridize to one or more preselected ribonucleic acids-of-interest such as preselected viral RNA. In these embodiments, the hybridization of the nanoswitch to the ribonucleic acid is considered a first binding interaction. Alternatively, a second binding interaction may be an additional binding interaction that occurs upon hybridization of a second nucleic acid such as to a second pair of detector strands. In embodiments, a single nanoswitch of the present disclosure may include 1-75 pairs of predetermined detector strands of the present disclosure, such as 2-50 pairs of detector strands, 5-30 pairs of detectors strands, 10-25 pairs of detector strands, or 20, 21, 22, 23, 24, 25, 26, 27 pairs of detector strands.
In embodiments, the nanoswitch is designed to detect one target ribonucleotide by hybridization of a first oligonucleotide and a second oligonucleotide, each having an overhang (i.e., a single-stranded region that is available for hybridization to the nucleic acid such as an oligo-ribonucleotide of interest). The first and second oligonucleotides, in this example, may be referred to as partially hybridized to the scaffold since each has a single-stranded overhang region and a region that is hybridized to the scaffold. The first and second oligonucleotides may be denoted as “detector 1” and “detector 2”. The overhangs may be referred to herein as 3′ overhangs and 5′ overhangs, referring to the directionality of the single-stranded region. The distance between the first and the second oligonucleotides, when bound to the scaffold, dictates the size of the loop and ultimately the migration distance of the nanoswitch when it is bound to the target (or when it is stabilized) via a latch binding interaction. In embodiments, the detector length may have a length of 5 to 30 or 7-20 nucleotides. In embodiments, the overhang segment of the detector may be about half the length of the total detector strand length.
In some embodiments, the first oligonucleotide and the second oligonucleotide, e.g., detector strands, are separated from each other by 100-6,000 nucleotides. In some embodiments, the first oligonucleotide and the second oligonucleotide are separated from each other by 500 to 5,000 nucleotides, 600-5,000 nucleotides, 1,000-5,000 nucleotides, or 1,000-3,000 nucleotides. In some embodiments, the first oligonucleotide and the second oligonucleotide are separated from each other by at least 500, 600, 700, 800, 900, 1,000, 1,500, 2,000, 2,500, 3,000, 3,500, 4,000, 4,500, 5,000, 5,500, 6,000, or more nucleotides. In some embodiments, the first oligonucleotide and the second oligonucleotide are located about equidistant about the center of the scaffold nucleic acid. In some embodiments, the first and second oligonucleotides bind to regions of the scaffold nucleic acid that are internal to the scaffold (i.e., such regions exclude the most 5′ and the most 3′ nucleotides of the scaffold).
In embodiments, such as when measured using gel electrophoresis, the open and closed nanoswitch conformations migrate differentially through a medium such as a gel medium. In embodiments, a circular scaffold such as circular M13 migrates the slowest, a linearized double-stranded version of M13 (without internal binding interactions) migrates fastest, and nanoswitches in looped conformations migrate in between. In embodiments, the migration distance differs based on the length of the loop. As an example, loops that are on the order of about 2590 base pairs are clearly distinguishable from loops that are on the order of about 600 base pairs. Loops of other sizes can also be distinguished from each other. The ability to distinguish between loops of different sizes means that the presence (or absence) of multiple targets (each detected by a complex having a loop of a particular size) can be determined simultaneously in a multiplexed assay. Such methods may be used to detect the presence of a single or multiple target and may form the basis of a diagnostic assay. Moreover, it should also be understood that nanoswitches having one loop can also be distinguished from nanoswitches having more than one loop, including those that have 2, 3 or more loops. In embodiments, a single type of nanoswitch can be used to detect two different targets and depending on the conformation of the nanoswitch (as determined by its migration distance in a gel), an end user can determine whether either or both targets are present in a sample. These nanoswitches can then also be extracted from the gel and the bound targets can be isolated.
In embodiments, electrophoresis is performed wherein a gel is run at 4 degrees Celsius to maintain the interaction of the targets to their binding partners (e.g., the binding of a protein target to target-specific antibodies) or to maintain latch binding interactions. It is contemplated that other separation medium is suitable for use herein such those used in capillary electrophoresis and liquid chromatography. In embodiments, reacting a first conformation of a nanoswitch, and when present, the second conformation of a nanoswitch with an indicator under conditions sufficient to form a signal refers to staining the one or more nanoswitches with a dye suitable for gel electrophoresis sufficient to fluoresce, glow or emit light as a signal.
In some embodiments, nanoswitches designed for DNA nanoswitch-ribonucleic acid complex formation are provided, such as those shown in
In embodiments, the 3′ overhang is not complementary to the 5′ overhang, and rather both the 3′ and the 5′ overhangs are complementary to a ribonucleic acid-of-interest such as viral RNA and suitable for forming a DNA nanoswitch-ribonucleic acid complex of the present disclosure. In embodiments, the entire ribonucleic acid-of-interest such as viral RNA (referred to as “Target “Key” or “oligonucleotide”) hybridizes to a combination of the 3′ and 5′ overhang. However, in embodiments, the method can also be performed in which the 3′ and 5′ overhangs are designed to hybridize only the 5′ and 3′ regions of a ribonucleic acid-of-interest, with the internal or middle region of the target ribonucleic acid remaining unhybridized. In this latter instance, the nanoswitch is designed to include a plurality of ribonucleic acids-of-interest differing sequences provided that they are at least complementary to the 3′ and 5′ overhangs. In embodiments, the nanoswitch detects non-adjacent sequences on the target. Such non-adjacent sequences may be separated by 1 or 2 nucleotides or by 10's or 100's of nucleotides, without limitation. In embodiments, ribonucleic acid-of-interest hybridized to the nanoswitch. In embodiments, the nanoswitch detects adjacent sequences on the target, depending upon the predetermined selection of overhang segments of the detector strands.
In embodiments, a nucleic acid structure of the present disclosure includes a DNA nanoswitch-ribonucleic acid complex including a deoxyribonucleic acid (DNA) nanoswitch and an oligonucleotide, wherein the DNA nanoswitch and oligonucleotide are configured to form a DNA-nanoswitch-ribonucleic acid complex having a second conformation characterized as locked, and a first conformation characterized as open in the absence of ribonucleotide such as viral RNA. In embodiments, the nanoswitch is configured such that the 3′ and 5′ overhangs come into sufficient proximity to each other in the presence of the ribonucleic acid-of-interest, and that it is only once the ribonucleic acid-of-interest hybridizes to the 3′ and 5′ overhangs that a looped conformation of a DNA-nanoswitch-ribonucleic acid complex is formed.
In some embodiments, the overhangs may be of different or identical lengths, relative to each other. The overhang length may range from 5-20 nucleotides in length, without limitation. The overhangs may have a length of 5 or more, or 6 or more, or 7 or more nucleotides. One or both overhangs may have a length of 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 nucleotides. The combined length of the overhangs may vary and may depend on their sequence and the length of the target nucleic acid. Their combined length may be 14 nucleotides or longer, without limitation. In some instances, the 3′ overhang and the 5′ overhang are of different lengths and their combined length is at least about 22 nucleotides.
In some embodiments, the combined length of the overhangs may be the same length as the ribonucleic acid-of-interest. Alternatively, the combined length of the overhangs may be longer or shorter than the length of the nucleic acid-of-interest. In some embodiments, the ribonucleic acid-of-interest may not bind to both overhangs to the same extent. In other words, one overhang may share more sequence complementarity with the ribonucleic acid-of-interest than the other overhang. In some embodiments, the overhangs will be referred to herein as the 3′ and 5′ overhangs intending the directionality of the overhangs. In some embodiments, the overhangs will be ligated to each other, as described herein, and thus the 3′ overhang may comprise a 3′ hydroxyl and the 5′ overhang may comprise a 5′ phosphate.
In some embodiments, the overhangs may be designed such that they include secondary structure such as but not limited to hairpin conformations. Such secondary structures may be melted during hybridization to the target, or they may be melted as a result of a change in condition or contact with an extrinsic trigger. Thus, also provided herein are compositions comprising any of the foregoing nucleic acid complexes. The composition may comprise a plurality of nucleic acid complexes. The nucleic acid complexes in the plurality may be identical to each other.
Alternatively, the nucleic acid complexes in the plurality may be different from each other. The nanoswitches may differ from each other with respect to their ribonucleic acid-of-interest specificity (e.g., the nucleotide sequence of their 3′ overhangs and/or the sequence of the 5′ overhangs). Nanoswitches may also differ from each other with respect to the distance between the 3′ overhang and the 5′ overhang along the length of the scaffold nucleic acid. Alternative embodiments for increasing signal are also contemplated herein and described below.
In embodiments, and as described above, one or more ribonucleic acids-of-interest may be pre-selected to hybridize to the one or more detectors and form a DNA-nanoswitch-ribonucleic acid complex of the present disclosure. In the embodiments, the one or more ribonucleic acids-of-interest may be an RNA, viral RNA, or a combination thereof. In embodiments, the ribonucleic acid-of-interest may be a naturally occurring ribonucleic acid, or one or more fragments thereof.
In embodiments, the ribonucleic acid-of-interest, as used herein, refers to the ribonucleic acid that is hybridized to the nanoswitch. It is to be understood that the ribonucleic acid-of-interest may derive from and thus be a fragment of a much larger ribonucleic acid such as for example viral RNA. Thus, a binding portion of the ribonucleic acid-of-interest (i.e., the ribonucleic acid bound to the nanoswitch) may range from about 7-50 nucleotides, or e.g., 10 to 35 nucleotides, or 5 to 30 nucleotides in some instances, while its parent ribonucleic acid may be much longer (for example on the order to kbs or more).
In some embodiments, the conditions that allow ribonucleic acid-of-interest such as viral RNA to hybridize to the 3′ overhang and the 5′ overhang may be standard hybridization conditions as known in the art. Such conditions may include a suitable concentration of salt(s) and optionally a buffer. The condition may also include EDTA in order to preserve the DNA nanoswitch-ribonucleic acid complex.
In some embodiments, the hybridization may be accomplished using a constant annealing temperature. Such constant temperature may range from about 4° C. to 55° C., 15° C. to 30° C., or 20° C. to 30° C., or may be about 25° C. The temperature may be regarded as room temperature (RT). The hybridization may be carried out over a period of hours such as 1, 2, 3, 4, 5 hours or more.
In some embodiments, the hybridization may be accomplished by decreasing the temperature from a temperature at which the ribonucleic acid-of-interest and the overhangs are not hybridized to each other to a temperature at which they are hybridized to each other. This is referred to herein as a temperature ramp or a decreasing annealing temperature. The starting temperature may be about 40-60° C. without limitation. The ending temperature may be about 4-25° C. without limitation. Thus, the temperature ramp may be from about 50° C. to about 4° C. or about 40° C. to about 4° C. In embodiments, a temperature ramp from about 46° C. to about 4° C. The change in temperature is typically carried out over 1-12 hours. Thus, the change in temperature may decrease by about 0.1-1° C. per minute.
Regardless of whether a constant or decreasing annealing temperature is used, the hybridization may also be carried out for much shorter periods of time, for example on the order of 10-30 minutes, provided readout can be achieved. Thus, in some instances, if the method determines if the nucleic acid-of-interest is present and has formed a DNA nanoswitch-ribonucleic acid complex of the present disclosure, then the hybridization period can be short, particularly if the ribonucleic acid-of-interest such as viral RNA is present in abundance. In some embodiments, longer hybridization times may be required. Similarly, if the ribonucleic acid-of-interest is present in low abundance, longer hybridization times may be required, particularly if an amplifying latch mechanism is used.
In some embodiments, only a portion or preselected portion of the ribonucleic acid-of-interest hybridizes to the nanoswitch of the present disclosure.
In embodiments, the biological sample is a sample that is being tested for the presence of one or more targets-of-interest such as viral RNA as described above. In embodiments, a target-of-interest may be present and thus contacted with a DNA nanoswitch of the present disclosure within in a mixture such as a biological sample or specimen. In embodiments, a target-of-interest may be disposed within a sample that may contain the target(s) or it may be suspected of containing the target(s). In embodiments, a sample may comprise non-target nucleic acid and be in the form of a mixture. Non-target nucleic acid, as used herein, refers to nucleic acids that are not the targets-of-interest or do not include a binding portion to the nanoswitch. In embodiments, the non-targets may be a peptide, protein or the like that does not react with or alter the conformation of the DNA nanoswitch or form a DNA nanoswitch-nucleic acid complex. The methods provided herein allow for the detection of a target such as a predetermined target even if such target is present in a molar excess of non-target nucleic acid. Thus, the sample may include on the order of micromolar quantities of non-target nucleic acid or protein and only nanomolar or picomolar quantities of target and still be able to detect the target. The target and non-targets may be present in the sample at a molar ratio of 1:102, 1:103, 1:104, 1:105, or up to 1:109.
In some embodiments, the sample may be derived from a biological sample such as a bodily fluid (e.g., a blood sample, a urine sample, a saliva sample, a sputum sample, a stool sample, a biopsy, and the like). The disclosure contemplates that such samples may be manipulated prior to contact or mixing with the nanoswitches of the present disclosure. For example, the samples may be treated to lyse cells, degrade or remove protein components, fragment nucleic acids such as genomic DNA, and the like.
In some instances, the target is or is derived from or is a fragment of viral RNA.
In embodiments, the methods of the present disclosure include detection of viral RNA. Such methods may be used to diagnose a condition, and thus may be referred to herein as diagnostic methods.
In embodiments, a method of the present disclosure includes contacting any of the foregoing DNA nanoswitch with viral RNA to form one or more DNA nanoswitch-ribonucleic acid complexes under conditions that allow a target, if present in the sample, to bind to the DNA nanoswitch and form a DNA nanoswitch-ribonucleic acid complex. In embodiments, the methods include detecting a conformation change between a DNA nanoswitch and a DNA nanoswitch-ribonucleic acid complex by moving the structures through a separation medium such as a gel medium. In embodiments, wherein a looped conformation is present within a medium such as an electrophoresis gel medium, the conformation is indicative of the presence of a specific target-of-interest such as a target viral RNA in the biological sample. In embodiments, in the presence of a target RNA, the DNA-nanoswitch-ribonucleic acid complex adopts a looped conformation as the DNA nanoswitch-target complex eliminates the linear conformation of the DNA nanoswitch.
In some embodiments, the conformation of the ribonucleic acid complex, e.g., DNA nanoswitch with viral RNA may be determined (or detected) using gel electrophoresis or liquid chromatography, or other separation technique. The gel electrophoresis may be a bufferless gel electrophoresis such as the E-Gel®. Agarose Gel Electrophoresis System (Life Technologies). In embodiments, methods may include detection of the target ribonucleic acid and detection and optionally purification or substantial purification of the target nucleic acid. In some embodiments, the method may also include measuring an absolute or relative amount of target ribonucleic acid. This can be done for example by measuring the intensity of bands on a gel or of fractions from a liquid chromatography separation.
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In embodiments, the present disclosure relates to a method of detecting a virus by reconfiguring a nucleic acid including: contacting a deoxyribonucleic acid (DNA) nanoswitch having a first conformation characterized as open with a biological specimen to form a mixture, wherein when the mixture comprises a ribonucleic acid-of-interest, the first conformation changes to a second conformation characterized as closed; processing the mixture under conditions sufficient to separate the first conformation, and when present, the second conformation; and reacting the first conformation, and when present, the second conformation with an indicator under conditions sufficient to form a signal. In some embodiments, the ribonucleic acid of interest is viral ribonucleic acid (viral RNA), long viral RNA, or one or more fragments thereof. In some embodiments, the ribonucleic acid-of-interest binds to the deoxyribonucleic acid (DNA) nanoswitch to form a second conformation including a loop. In some embodiments, the biological specimen comprises one or more ribonucleic acids-of-interest. In some embodiments, the one or more ribonucleic acid-of-interest include RNA or a fragment thereof from ZIKA, EBOLA, SARS, or SARS-CoV-2, or variants thereof. In some embodiments, the ribonucleic acid-of-interest is SEQ ID NO:1, or a fragment thereof. In some embodiments, the ribonucleic acid-of-interest is SARA-CoV-2 RNA, or a fragment thereof as described in U.S. Pat. No. 10,815,539 (herein entirely incorporated by reference). In some embodiments, processing the mixture includes electrophoresing the mixture under conditions sufficient to separate the first conformation and the second conformation. In some embodiments, a formation of the second conformation signals a presence of one or more ribonucleic acids. In some embodiments, the second conformation has an altered functionality compared to the first conformation. In some embodiments, the first conformation is configured to change to a second conformation when contacted with ribonucleic acid-of-interest, and wherein the second conformation is configured to report a ribonucleic acid-of-interest.
In some embodiments, the present disclosure relates to a method of reconfiguring a nucleic acid including: contacting a deoxyribonucleic acid (DNA) nanoswitch having a first conformation, with a ribonucleic acid to form a DNA nanoswitch-nucleic acid complex having a second conformation, wherein the second conformation is characterized as locked; processing a mixture under conditions sufficient to separate the first conformation and the second conformation; and contacting the first conformation and second conformation with an indicator under conditions sufficient to form a signal. In some embodiments, the signal is predetermined to show a presence or absence of ribonucleic acid.
In some embodiments, the present disclosure relates to a nucleic acid structure or molecule, including: a DNA nanoswitch-nucleic acid complex including a deoxyribonucleic acid (DNA) nanoswitch and a ribonucleic acid binding site, wherein the DNA nanoswitch has a first conformation characterized as open, and a second conformation characterized as closed when in a presence of ribonucleic acid.
In some embodiments, the present disclosure relates to a nucleic acid structure or molecule, including: a DNA nanoswitch-nucleic acid complex including a deoxyribonucleic acid (DNA) nanoswitch and an RNA binding site, wherein the DNA nanoswitch has a first conformation characterized as open, and a second conformation characterized as closed when in a presence of RNA bound to the RNA binding site.
Referring to
In embodiments, the present disclosure relates to one or more (such as a plurality) of DNA nanoswitches which individually include a plurality of RNA binding sites configured to target different segments of a long (viral RNA). In embodiments, such as where a DNA nanoswitch is configured to have a multi-targeting approach by the inclusion of a plurality of RNA binding sites within the DNA nanoswitch, wherein each RNA binding site may be preselected to bind to a separate region or segment of viral RNA-of-interest, such embodiments, have the advantage of increasing the signal of the DNA nanoswitches subjected to gel electrophoresis. For example, embodiments, of the present disclosure may include a method of detecting an RNA virus by reconfiguring a plurality of nucleic acid including: contacting a plurality of deoxyribonucleic acid (DNA) nanoswitches having a first conformation characterized as open with a biological specimen to form a mixture, wherein the mixture includes a plurality of RNA segments from a single long ribonucleic acid-of-interest, the first conformation changes to a second conformation characterized as closed; processing the mixture under conditions sufficient to separate the first conformation, and when present, the second conformation; and reacting the first conformation, and when present, the second conformation with an indicator under conditions sufficient to form a signal. In embodiments, the single long viral ribonucleic acid-of-interest is viral ribonucleic acid (viral RNA) characterized as long such as e.g., a viral RNA including nucleotides in an amount greater than 5,000 nucleotides, 6,000 nucleotides, 7,000 nucleotides, 8,000 nucleotides, 9,000 nucleotides, or greater than 10,000 nucleotides. In embodiments, the single long ribonucleic acid-of-interest is not fragmented and/or is fragmented into a plurality of RNA segments. In embodiments, the plurality of deoxyribonucleic acid (DNA) nanoswitches include DNA nanoswitches including a plurality of different predetermined RNA binding sites, configured to bind to different predetermined segments of a fragmented long viral RNA or unfragmented long viral RNA. For example, a first DNA nanoswitch may be configured to change conformation when contacted with a first, second, or third predetermined RNA sequence or segment at a first, second, or third RNA binding site of the DNA nanoswitch. In embodiments, a single DNA nanoswitch of the present disclosure may be configured to change conformation (such as to a loop) when contacted with 3-100, or 10-50 different sequences or segments of unfragmented or fragmented long viral RNA. In embodiments, a multi-targeting approach where a single DNA nanoswitch may react with a plurality of predetermined RNA sequences such as those of a long RNA, or those derived from a long RNA may increase the signal of the present disclosure. By increasing the signal, the methods of the present disclosure are highly sensitive and robust.
In embodiments, the present disclosure relates to one or more (such as a plurality) of DNA nanoswitches which individually or collectively include a plurality of variant RNA binding sites. Here the DNA nanoswitches may be configured to target the same segment of a long (viral RNA). In embodiments, such as where a DNA nanoswitch is configured to include a plurality of variant RNA binding sites a single-targeting approach by the inclusion of a plurality of variant target regions within the DNA nanoswitch may have the advantage of improving binding kinetics between the variant RNA binding sites of the DNA nanoswitch and the RNA-of-interest. In embodiments, signal formation may be promoted for such DNA nanoswitches subjected to gel electrophoresis. For example, embodiments of the present disclosure may include a method of detecting an RNA virus by reconfiguring a plurality of nucleic acid structures including: contacting a plurality of deoxyribonucleic acid (DNA) nanoswitches having a first conformation characterized as open with a biological specimen to form a mixture, wherein the mixture includes an RNA of interest such as from a single long ribonucleic acid-of-interest, the first conformation changes to a second conformation characterized as closed; processing the mixture under conditions sufficient to separate the first conformation, and when present, the second conformation; and reacting the first conformation, and when present, the second conformation with an indicator under conditions sufficient to form a signal. In embodiments, the DNA nanoswitch may be include one or more RNA binding sites characterized as a variant binding sites. As used herein the variant target region may vary between different DNA nanoswitches. The RNA binding sites variants may be highly related sequences, or highly homologous sequences having 90%, 95%, 97%, 99% sequence identity. The variant RNA binding sites may also bind to the same region of target RNA under substantially similar hybridization conditions. The variants may have different lengths, such that one variant appears truncated upon comparison to a highly related variant.
Embodiments, of the present disclosure further include embodiments as described herein alone, or in combination. For example, it is possible to mix and match the embodiments described herein. In some embodiments, the methods of the present disclosure are preselected to obtain a maximum signal. For example, it is possible to use a plurality of different multi-targeting DNA nanoswitches to target a plurality of regions on a viral RNA-of-interest. In embodiments, 1-5 different multiple (1-25) target nanoswitches may be used to target over 100 regions on SARA-CoV-2 viral RNA, which can give a 120 fold benefit in limit of detection. In embodiments, the techniques and DNA nanoswitches are expandable up to about 50 targets per nanoswitch, and about 10-20 nanoswitches targeting about 1,000 regions with about 1000-fold increase in sensitivity.
Referring now to
In embodiments, the present disclosure includes a kit, including: one or more DNA nanoswitches of the present disclosure, wherein, when present, the DNA nanoswitch hybridizes a viral RNA or a fragment thereof; and a separation medium such as a gel strip. In embodiments, the kit further includes a buffer.
Detection of viruses is critical for controlling disease spread and for informing clinical interventions. Recent emerging viral threats including Zika virus, Ebola virus, and SARS-Cov-2 (responsible for the COVID-19 outbreak) highlight the cost and difficulty in responding rapidly. To address these challenges, the present disclosure provides a platform for low-cost and rapid detection of viral RNA with DNA nanoswitches designed to mechanically reconfigure in response to specific viruses. Using Zika virus as a model system, non-enzymatic detection of viral RNA is shown to the attomole level, with selective and multiplexed detection between related viruses and viral strains. For clinical-level sensitivity in biological fluids, the assay was paired with a sample preparation step using either RNA extraction or isothermal pre-amplification. The assays of the present disclosure can be performed with minimal or no lab infrastructure, and are readily adaptable to detect other RNA viruses. The adaptability of the methods of the present disclosure was demonstrated by quickly developing and testing DNA nanoswitches for detecting a fragment of SARS-CoV-2 RNA in human saliva. Given this versatility, field implementation will improve the ability to detect emergent viral threats and ultimately limit their impact.
Oligonucleotides were purchased from Integrated DNA Technologies (IDT) with standard desalting, and the full sequences of all strands is listed in the tables below (Tables 1 to 10).
AGTGTCTGATTGC (SEQ ID NO: 2)
TTGTCAAGGTAGGCTTCAACCGATTGAGGGAGGGAAGGTA
(SEQ ID NO: 5)
TCAACCGATTGAGGGAGGGA
(SEQ ID NO: 7)
TCAACCGATTGAGGGAGGGAA
(SEQ ID NO: 9)
TCAACCGATTGAGGGAGGGAAG
(SEQ ID NO: 11)
TCAACCGATTGAGGGAGGGAAGG
(SEQ ID NO: 13)
TCAACCGATTGAGGGAGGGAAGGT
(SEQ ID NO: 15)
TCAACCGATTGAGGGAGGGAAGGTA
ATGGTGGCATCACACTCAACCGATTGA
CAGCCTCTGTCCACTTCAACCGATTGA
GGTGAATTGGGCGTTTCAACCGATTGA
CCTTCTTGACTCCCTTCAACCGATTGA
GAGTCAGGATGGTACTCAACCGATTG
ATCTCCACATGATGTTCAACCGATTGA
CATGTGTCGTTCTTCTCAACCGATTGA
GGGGTGTCCAATTAGTCAACCGATTGA
AGCCGGGGACTGTTTTCAACCGATTGA
CGCTCTCTCTCCGGGTCAACCGATTGA
CCTGCTGGGTAGTCCTCAACCGATTGA
CCGGGAATGCGTCACTCAACCGATTG
TGTTTTCTGGAACTCTCAACCGATTGA
CCTTCCCATTAGATGTCAACCGATTGA
ATGGCTATTCCTGTGTCAACCGATTGA
TCTCTCTCCCCATCCTCAACCGATTGA
CTCTAGAACTTCCTCTCAACCGATTGA
GGTTTTTTGACTCAGTCAACCGATTGA
GCAGTCCTCAGTCACTCAACCGATTGA
GCTCTGGGAGGAGAGTCAACCGATTG
ATCGTCCAGTGAGCATCAACCGATTGA
CTTGGAGTGCTGGCCTCAACCGATTGA
TTCCCTTCTGGTGTGTCAACCGATTGA
GCTCGTAAGTGGCTTTCAACCGATTGA
TCCACTACGCCATGCTCAACCGATTGA
TTGAGCTGTAGCTTGTCAACCGATTGA
CTGAGGAAATGGGTCTCAACCGATTG
GCGTTTCTTGCCTTCTCAACCGATTGA
CGGTTCTTGGGTTCTTCAACCGATTGA
TCTTGGTGTTGGTCTTCAACCGATTGA
TTCCCAGCAACCCCA (SEQ ID NO: 120)
AAAGCATGATAGTCTTCAACCGATTGAGGGAGGGAAGG
TTGTTCGGTATGGGTAAAGGGATGCCATTC (SEQ ID
GAATGGCATCCCTTT (SEQ ID NO: 123)
ACCCATACCGAACAATCAACCGATTGAGGGAGGGAAGG
TGGTGAATTGGGCGT (SEQ ID NO: 126)
TATCTCAACCTTCGCTCAACCGATTGAGGGAGGGAAGG
GGATCTTAGTGAATG (SEQ ID NO: 129)
TGAACGCTGCGGTACTCAACCGATTGAGGGAGGGAAGG
CGGCTGGGGTAATGA (SEQ ID NO: 132)
AAGTTGTCAGAGCAGTCAACCGATTGAGGGAGGGAAGG
TTCCCAGCAAACCTA (SEQ ID NO: 135)
AGAGCATAATGGTCTGTTTTAGCGAACCTCCCGACTTGC
CTGTTTGGCATGGGCAAAGGGATGCCATTT (SEQ ID
AAATGGCATCCCTTT (SEQ ID NO: 138)
GCCCATGCCAAACAGGTTTTAGCGAACCTCCCGACTTG
TGGTGAATTAGGCGT (SEQ ID NO: 141)
AACCTCGACTTTCGCGTTTTAGCGAACCTCCCGACTTGC
GGACCTTGGTGAATG (SEQ ID NO: 144)
TGAATGCCGCAGTGCGTTTTAGCGAACCTCCCGACTTG
CAGCTGGGGTGATGA (SEQ ID NO: 147)
GAGTTGTCAATGCGGGTTTTAGCGAACCTCCCGACTTG
AATGCTGTCAGTTCATGGCTCCCAGCACAGTGG
GATGATCGTTAATGATACAGGACATGAAACTGAT
TCACCAAGAGCCGAAGCCACCCTGGGGGGTTTT
AG (SEQ ID NO: 150)
AATTCTAATACGACTCACTATAGGGAGAAGGAA
GGTGAATTGGGCGTTTCAACCGATTGAGGGAGG
TCATTAACGATCATCTCAACCGATTGAGGGAGG
AATTCTAATACGACTCACTATAGGGAGAAGGTG
CAATACAGATCATGGTCAACCGATTGAGGGAGG
Table 11 (variable oligos) In embodiments, suitable polynucleotides include those having a nucleotide sequence having at least 80%, 90%, 95%, 97%, 99% sequence identity to SEQ ID NOS: 278 to 289 as set forth in the table below.
AGTAATAACATCAC (SEQ ID NO: 281)
ATTCGACAACTCGTATTAAATCCTTTGCCCGAACGTTATTAATTT
TGGGTTATATAACTATATGTAAATGCTGATGCAAATCCAATCGCA
GTTTTAGCGAACCTCCCGACTTGCGGGAGGTTTTGAAGCCTTAA
TCAACCGATTGAGGGAGGGAAGGTAAATATTGACGGAAATTATT
Table 12 depicts filler oligonucleotides as described herein.
In embodiments, suitable polynucleotides include those having a nucleotide sequence having at least 80%, 90%, 95%, 97%, 99% sequence identity to SEQ ID NOS: 290 to 297 as set forth in the tables above.
Nanoswitches were constructed as described previously (See e.g, M. A. Koussa, et al., DNA nanoswitches: a quantitative platform for gel-based biomolecular interaction analysis. Nature Methods. 12, 123 (2015); A. R. Chandrasekaran, et al., Cellular microRNA detection with miRacles: microRNA-activated conditional looping of engineered switches. Science Advances. 5, eaau9443 (2019)), and/or as described in U.S. Patent Publication No. 2018/0223344 (all of which are herein entirely incorporated by reference). In an embodiment, a genomic single-stranded DNA (New England Biolabs M13mp18) was linearized using targeted cleavage with BtsCl restriction enzyme. The linearized single-stranded DNA was then mixed with a molar excess of an oligonucleotide mixture containing backbone oligos, detectors, and annealed from 90° C. to 20° C. at 1° C. min−1 in a T100™ Thermal Cycler (Bio-Rad, USA). Following construction, the nanoswitches were purified using liquid chromatography (LC) purification (See e.g., K. Halvorsen, et al., Shear Dependent LC Purification of an Engineered DNA Nanoswitch and Implications for DNA Origami. Anal. Chem. 89, 5673-5677 (2017)) to remove excess oligonucleotides. The concentration of purified nanoswitches were determined by measuring A260 absorbance with a Thermo Scientific NanoDrop 2000.
Plasmids containing the full-length ZIKV (Cambodia FSS13025 strain; pFLZIKV) and DENV-2 (strain 16681, pD2/IC-30P) cDNAs were provided. pFLZIKV was linearized with ClaI (New England Biolabs, NEB), and pD2/IC-30P was linearized with XbaI (NEB). Digested plasmids were extracted with phenol:chloroform:isoamyl alcohol and then precipitated. Linearized plasmids were in vitro transcribed (Thermo Fisher Scientific) and the resulting viral RNA cleaned by MEGAclear Transcription Clean-Up Kit (Thermo Fisher Scientific). The protocols of these two kits were followed except that the purification column was not heated in the elution step of the viral RNA because high temperature can result in degradation of the viral RNA.
Viral RNA was fragmented by using 10× Fragmentation buffer (NEB) and the recommended protocol. Briefly, the ZIKV RNA obtained from in vitro transcription was mixed with fragmentation buffer (1× final) and then incubated at 94° C. in a thermal cycler for 1, 3, 6, or 9 min. RNA fragmentation analyzer (Agilent, model 5003) was used to quantify the length distribution of RNA fragments by using the DNF-471 Standard Sensitivity RNA Analysis Kit (
Human hepatocarcinoma (Huh7) cells were maintained in Dulbecco's modified Eagle's medium (DMEM) (Life Technologies) supplemented with 10% fetal bovine serum (FBS, VWR Life Science Seradigm), 10 mM non-essential amino acids (NEAA; Life Technologies), and 5 mM L-glutamine (Life Technologies). Cells were passaged once every three days and maintained at 37° C. with 5% CO2. Twenty-four hours prior to infection Huh7 cells were seeded into tissue culture plates. The following day, one plate was counted. The other two plates were used for mock- and ZIKV-infection, where cells were infected at a multiplicity of infection of one. The original Cambodia and Uganda (MR766) ZIKV stocks were provided. To isolate RNA from mock- and ZIKV-infected cells, media from the cells was aspirated and then the cell monolayer was washed once with ice-cold PBS. Hereafter, the cells in each tissue culture plate were lysed in 1 mL TRIzol (Invitrogen) and total RNA extracted per the manufacturer's instructions.
For experiments using ZIKV infectious particles in PBS/urine (
The total detection sample volume was 10 μl with 10 mM MgCl2, 1×PBS, nanoswitch at 100 pM final concentration. Samples were incubated in a thermal cycler with thermal annealing from 40° C. to 25° C. at 1° C. min−1 or room temperature (e.g. the NASBA related detections). Before loading into the gel, the samples were stained by GelRed (Biotium Inc.) at 1× concentration (or 3.3× for total RNA detection) and mixed with 2 μl 6× loading dye (15% Ficoll with 6.6% of a saturated bromophenol blue solution in water).
For the experiment in
Detection of Viral RNA from Total RNA
First, 500 ng total RNA extracted from uninfected/infected cells was fragmented at 94° C. for 9 minutes in 1× fragmentation buffer. Fragmented total RNA was then mixed with nanoswitches (100 pM, MgCl2 (10 mM) and PBS (1×), and the mixture was made up to 10 μl with nuclease-free water. Samples were then incubated in a thermal annealing ramp from 40° C. to 25° C. over ˜12 hours (at −0.1° C./cycle and 5 min for each cycle, for a total of 150 cycles). After the incubation, samples were stained with GelRed at 3.3× concentration and incubated at room temperature for 30 min. Before loading the gel, 2 μl of 6× blue loading dye was mixed with each sample, and 10 μl sample was loaded to each well. Samples were run in a 0.8% agarose gel at 65-75 V for about 70-90 minutes in the cold room.
Detection of Viral RNA Extracted from Urine
For the detection of viral RNA extracted from urine, DNA/RNA shield buffer was added (included with the Quick-RNA Viral Kit from ZYMO research) into urine and then mixed in the RNA with blocking oligos (200 nM) into 200 μl human urine (purchased from Innovative Research, Inc.) to mimic a clinical sample, and performed the RNA extraction immediately. Then, Quick-RNA Viral Kit (Zymo research) was used to extract the viral RNA from the urine. After RNA extraction, RNase Inhibitor was added (final concentration, 1 U/μl) to the solution. Different amounts of ZIKV RNA were tested (
First, the classic NASBA protocol (34) was employed to prove the concept (
For the ZIKV related NASBA experiments, the ZIKV infectious particles were spiked (starting at 1180 pfu/μl) into 1×PBS or 10% human urine (purchased from Innovative Research, Inc.) to concentrations of 897, 200, and 20 pfu/μl. Subsequently, blocking oligo (200 nM) was added and the viral RNA was released by heating the samples at 94° C. for 3 minutes within 10 ul volume. For the human urine samples, RNase Inhibitor (NEB, Murine) was also added at a concentration of 2 U/μl before heating. After cooling down to room temperature, 0.5 μl RNase Inhibitor at 40 U/μl (NEB, Murine) was added to 10 μl human urine sample to protect the viral RNA. Total volume of each NASBA reaction was scaled down to 6 μl which contains 1.25 μl Enzyme COCKTAIL (NEC 1-24), 2 μl 3× buffer (NECB-24), 0.48 μl NTPs mix at 25 mM, 0.3 μl dNTPs mix at 20 mM, 0.2 μl two primers mix at 10 pM, 0.2 μl RNase Inhibitor with 40 U/μl (NEB, Murine) and 1.57 μl of viral RNA. The sample was incubated at 41° C. for 2 hours in the thermal cycler and followed by heating at 94° C. for 5 minutes to deactivate all enzymes. Then 1 μl of the NASBA sample was used in the following DNA nanoswitch detection assay in PCR tubes with 10 μl final volumes. The assay was finished by incubating at room temperature for two hours. After mixing with GelRed at 1× concentration and 2 μl 6× blue loading dye, the detection samples were loaded to the 25 ml 0.8% agarose gel which was run in 0.5×TBE buffer at 75 V at room temperature for 45 minutes.
A gBlock gene fragment of the SARS-CoV-2 RNA segment (56) was purchased from IDT (Table 9). Then, PCR amplification (Qiagen, Taq PCR Core Kit) was used to create more copies with a T7 promoter that was added to the 5′ end of the forward primer. Afterword, dsDNA template was cleaned by the QIAquick PCR Purification Kit (Qiagen). Finally, the SARS-CoV-2 RNA was obtained by in vitro transcription (NEB, HiScribe™ T7 High Yield RNA Synthesis Kit) and cleaned by MEGAclear Transcription Clean-Up Kit (Thermo Fisher Scientific). RT-PCR detection was performed using the Luna Universal One-Step RT-qPCR kit from NEB and following its protocol. 1.5 μl RNA sample was used in 20 μl reaction mix.
For preparation of human saliva sample, pooled human saliva (purchased from Lee Biosolutions, Inc) was heated at 94° C. for 3 min to mimic the process of destroying viral capsid to release the RNA. Then, the SARS-CoV-2 RNA was spiked into diluted saliva (10%) with blocking oligos (˜200 nM) and RNase Inhibitor (2 U/μl). For the NASBA-based detection of SARS-CoV-2 RNA purchased from Twist Biosciences, NASBA kits purchased from Life Sciences Advanced Technologies Inc. were used. Briefly, 3.3 μl 3× buffer (NECB-24), 1.7 μl 6× Nucleotide Mix, 0.4 μl two primers mix at 10 μM, 0.25 μl RNase Inhibitor with 40 U/μl (NEB, Murine) and 2 μl viral RNA sample with different concentrations were mixed first and heated at 65° C. for 2 min and then the samples were incubated at 41° C. and 2.5 μl Enzyme COCKTAIL (NEC 1-24) was mixed. The samples were incubated at 41° C. for 40 min in the thermal cycler followed by heating at 94° C. for 5 minutes to deactivate all enzymes.
Then 1 μl of the NASBA amplified RNA sample was used in the following DNA nanoswitch detection assay in PCR tubes with 10 μl final volumes. Two nanoswitches were developed and used to target the amplified RNA pieces on two different regions (
The detection samples were run in 25 ml 0.8% agarose gels unless otherwise noted, cast from molecular biology grade agarose (Fisher BioReagents) dissolved in 0.5×TBE buffer. Typical running conditions were 75 V for 45 to 70 minutes at room temperature or cold room. Samples were mixed with a Ficoll-based blue loading dye prior to loading. Imaging was completed on a Bio-Rad Gel Doc XR+ imager with different exposure times based on the brightness of the detection bands. The detection efficiency was analyzed using included Image Lab software (
Embodiments for detecting the presence of viral RNA are based on using DNA nanoswitches that have been designed to undergo a conformational change (from linear to looped) upon binding a target viral RNA (
In some embodiments, an example of test conditions may include: preparing and purifying nanoswitches; using 1-2 uL of nanoswitch at 250-1000 pM in a 10 uL reaction with test reagent, final buffer of 20 mM Tris-HCl pH 8 and 30 mM MgCl2; reacting the mixture at 40° C. for 20 minutes-2 hours; adding 1 uL of 300 mM EDTA; adding 1 uL of a 10× GelRed dye solution and 2 uL of a 6× loading dye; loading 10 uL of solution into a 0.8% unstained agarose gel; and running the gel electrophoresis at 75V (for a OWL B1A minigel) for 20-45 minutes.
As a first proof-of-concept for detecting ZIKV, DNA nanoswitches were designed to target an already validated sequence in the ZIKV genome that has been used to bind primers in qPCR (22) (all oligo sequences are specified in Tables 1 to 10). DNA nanoswitches were made by hybridizing single-stranded DNA (ssDNA) oligos to linearized single-stranded M13mp18 (M13) genomic DNA in a thermal annealing ramp for 1 hour (See M. A. Koussa, K. Halvorsen, A. Ward, W. P. Wong, DNA nanoswitches: a quantitative platform for gel-based biomolecular interaction analysis. Nature Methods. 12, 123 (2015)) and purified them by high-performance liquid chromatography (HPLC) (23). For an initial detection target, in vitro transcribed RNA from the pFLZIKV infectious plasmid containing the full length genome of the Cambodia ZIKV isolate (FSS13025) was used (see e.g.,
Having shown successful detection of ZIKV RNA using a single target sequence, it is possible to exploit the large genome size (˜11,000 nucleotides) to increase detection signal through multiple targets. Once the long viral RNA is fragmented, the number of available target sequences increases dramatically. Since the detection signal is proportional to the number of looped nanoswitches, a nanoswitch mixture for different target sequences within the viral genome is expected to provide an increased signal. To test this, an algorithm for choosing multiple sequence regions in the viral genome was developed that can be targeted by the nanoswitches. First, the default target length as 30 nucleotides was selected based on results from screening nanoswitches with different detection arm lengths (
The quality and function of each nanoswitch in the panel of 18 nanoswitches was validated. All nanoswitches performed well with a molar excess of positive DNA controls (20:1 DNA control to nanoswitch), although they showed more signal variation with fragmented ZIKV RNA (
In embodiments, high sensitivity is one of the key requirements for virus detection. Clinical levels of ZIKV RNA in body fluids of infected patients are often in the femtomolar range (See e.g., D. Musso, D. J. Gubler, Zika Virus. Clinical Microbiology Reviews. 29, 487-524 (2016); K. Pardee, et al., Rapid, Low-Cost Detection of Zika Virus Using Programmable Biomolecular Components. Cell. 165, 1255-1266 (2016); and A.-C. Gourinat, O. O'Connor, E. Calvez, C. Goarant, M. Dupont-Rouzeyrol, Detection of Zika Virus in Urine. Emerg Infect Dis. 21, 84-86 (2015)) making amplification a prerequisite for most detection approaches. Based on observation that DNA nanoswitches can detect microRNAs (˜22 nucleotides) in the sub-picomolar scale (See A. R. Chandrasekaran, M. MacIsaac, P. Dey, O. Levchenko, L. Zhou, M. Andres, B. K. Dey, K. Halvorsen, Cellular microRNA detection with miRacles: microRNA-activated conditional looping of engineered switches. Science Advances. 5, eaau9443 (2019)) without amplification, it was desired to assess the sensitivity of the methods of the present disclosure for ZIKV RNA detection. The DNA nanoswitch mixture was reacted with different amounts of fragmented RNA in a 12-hour annealing temperature ramp from 40° C. to 25° C. The results showed visible detection for ZIKV RNA as low as 12.5 pg (˜3.5 attomole or ˜2.1×106 copies) in a 10 μl reaction volume (
Another key requirement for a clinical virus detection assay is specificity. Since ZIKV and Dengue virus (DENV) have overlapping geographical distributions and clinical symptoms, infection with either virus may result in clinical misdiagnosis (See E. S. Paixão, M. G. Teixeira, L. C. Rodrigues, Zika, chikungunya and dengue: the causes and threats of new and re-emerging arboviral diseases. BMJ Global Health. 3, e000530 (2018)). Serological diagnostic assays are known to show antibody cross-reactivity between the two viruses, and DENV has some similarity to ZIKV in its envelope protein (See W. Dejnirattisai, P. Supasa, W. Wongwiwat, A. Rouvinski, G. Barba-Spaeth, T. Duangchinda, A. Sakuntabhai, V.-M. Cao-Lormeau, P. Malasit, F. A. Rey, J. Mongkolsapaya, G. R. Screaton, Dengue virus sero-cross-reactivity drives antibody-dependent enhancement of infection with zika virus. Nature Immunology. 17, 1102-1108 (2016)) and genome sequence (See e.g., K. Pardee, et al., Rapid, Low-Cost Detection of Zika Virus Using Programmable Biomolecular Components. Cell. 165, 1255-1266 (2016); and R. G. Huber, et al., Structure mapping of dengue and Zika viruses reveals functional long-range interactions. Nat Commun. 10, 1-13 (2019)). To test the specificity of this approach, a similar panel of nanoswitches was designed to detect DENV (
Further applying the techniques of the present disclosure to detect ZIKV RNA in biological samples, Huh7 cells were either mock-infected or infected with the Cambodia ZIKV strain at a multiplicity of infection of 1 and extracted RNA from the ZIKV infected cells at 1-, 2- and 3-days post-infection (See e.g., G. Bonenfant, N. Williams, R. Netzband, M. C. Schwarz, M. J. Evans, C. T. Pager, Zika Virus Subverts Stress Granules to Promote and Restrict Viral Gene Expression. Journal of Virology, 93, e00520-19 (2019)). The nanoswitch assays of the present disclosure detected ZIKV viral RNA from the infected cells but not the mock infected cells (See e.g.,
Moving toward clinical applications, detection of relevant levels of ZIKV RNA from biological fluids is demonstrated. ZIKV is present in the serum, urine, and other body fluids of infected patients (See e.g., G. Paz-Bailey, et al., Persistence of Zika Virus in Body Fluids—Final Report. New England Journal of Medicine, 379, 1234-1243 (2018). The viral loads can vary dramatically between individuals, body fluid, and post infection time (See V. M. Corman, et al., Assay optimization for molecular detection of Zika virus. Bull World Health Organ. 94, 880-892 (2016); and D. Musso, D. J. Gubler, Zika Virus. Clinical Microbiology Reviews. 29, 487-524) (2016)), but are frequently in the sub-femtomolar to femtomolar range, with ZIKV in human urine reported as high as 220×106 copies/ml (365 fM) (26). While the nanoswitch sensitivity for in vitro transcribed viral RNA in buffer approaches clinically relevant concentrations, detection from body fluids is further challenged by varying viral loads and by body fluids that can reduce the performance of the nanoswitches due to physiological conditions and nuclease activity (See e.g., A. R. Chandrasekaran, J. Zavala, K. Halvorsen, Programmable DNA Nanoswitches for Detection of Nucleic Acid Sequences. ACS Sens. 1, 120-123 (2016); and C. H. Hansen, et al., Nanoswitch-linked immunosorbent assay (NLISA) for fast, sensitive, and specific protein detection. PNAS. 114, 10367-10372 (2017)). To overcome these potential difficulties, two independent solutions were investigated: 1) adding a pre-processing step to extract RNA from body fluids such as urine, or 2) adding an isothermal pre-amplification step. In the first approach, a clinically relevant amount of in vitro transcribed ZIKV RNA was spiked into human urine and processed viral RNA extraction using a commercial RNA extraction kit. RNase inhibitors were included to minimize RNA degradation in urine. The extracted RNA was then mixed with the nanoswitches and demonstrated non-enzymatic, clinical level detection of the RNA at 1.7×105 copies/μl (0.28 pM) (See e.g.,
With the emerging outbreak of SARS-CoV-2 in January 2020, the DNA nanoswitches were tested against the new virus. Following a similar strategy as for ZIKV, a target region was identified, nanoswitches were developed, and used the NASBA strategy to detect a SARS-CoV-2 RNA in 10% human saliva. Following the ZIKV protocol detection was validated for an in vitro transcript of a short segment of SARS-CoV-2 RNA in ˜5 hours, and cross validated with RT-PCR (
Taken together, programmable DNA nanoswitches are suitable for a robust viral RNA detection platform, that is readily adaptable as shown in the detection of SARS-CoV-2. The platform has key advantages over existing methodologies in terms of selectivity and specificity, as shown in the experiments with ZIKV and closely related DENV, as well as two closely related ZIKV strains. Moreover, DNA nanoswitch viral RNA detection strategy has femtomolar detection limit without an RNA amplification step, and attomolar detection limit when used with amplification. These limits are within a clinically relevant range and therefore the DNA nanoswitch assay together with the bufferless gel cartridge presents a putative diagnostic assay for clinical detection of RNA viruses in low resource areas without significant laboratory infrastructure.
The functionality of the DNA nanoswitches of the present disclosure is largely enabled by DNA nanotechnology, which has become a well-established field that uses DNA as a functional material to fabricate nanostructures (See e.g., N. C. Seeman, DNA in a material world. Nature. 421, 427-431 (2003)). Biosensing is a particularly promising application of DNA nanotechnology (See e.g., M. Xiao, W. Lai, T. Man, B. Chang, L. Li, A. R. Chandrasekaran, H. Pei, Rationally Engineered Nucleic Acid Architectures for Biosensing Applications. Chem. Rev. 119, 11631-11717 (2019)), and reconfigurable DNA devices (See e.g., L. Zhou, A. E. Marras, C.-M. Huang, C. E. Castro, H.-J. Su, Paper Origami-Inspired Design and Actuation of DNA Nanomachines with Complex Motions. Small. 14, e1802580 (2018)) have been demonstrated for the detection of DNA, RNA, proteins, and pH. (See, e.g., L. Zhou, et al., Paper Origami-Inspired Design and Actuation of DNA Nanomachines with Complex Motions. Small. 14, e1802580 (2018); T. Funck, et al., Sensing Picomolar Concentrations of RNA Using Switchable Plasmonic Chirality. Angewandte Chemie. 130, 13683-13686 (2018); S. M. Douglas, et al., A Logic-Gated Nanorobot for Targeted Transport of Molecular Payloads. Science. 335, 831-834 (2012); and S. Modi, et al., A DNA nanomachine that maps spatial and temporal pH changes inside living cells. Nature Nanotechnology. 4, 325 (2009)). However, most designs are complex and require laborious readout with advanced microscopy that reduces their practicality. A few approaches have overcome this practicality hurdle to provide widely useful solutions to problems in biological imaging (e.g. DNA-PAINT in super-resolution microscopy (See e.g., R. Jungmann, et al., Multiplexed 3D cellular super-resolution imaging with DNA-PAINT and Exchange-PAINT. Nature methods. 11, 313-318 (2014)) and DNA scaffolds for NMR and cryo-EM and biosensing (e.g. detection of lysosomal disorders and mapping cellular endocytic pathways). (See e.g., M. J. Berardi, et al., Mitochondrial uncoupling protein 2 structure determined by NMR molecular fragment searching. Nature. 476, 109-113 (2011); T. G. Martin, et al., Design of a molecular support for cryo-EM structure determination. PNAS. 113, E7456-E7463 (2016); K. Leung, et al., A DNA nanomachine chemically resolves lysosomes in live cells. Nature Nanotechnology, 1 (2018); and S. Modi, et al., Two DNA nanomachines map pH changes along intersecting endocytic pathways inside the same cell. Nature Nanotech. 8, 459-467 (2013)). The DNA nanoswitches take a reductionist approach, resulting in assays that are robust and sensitive, yet simple to adapt and do not require multiple steps or expensive equipment. With this work, RNA virus detection is added to the existing suite of DNA nanoswitch assays that already includes protein (See e.g., C. H. Hansen, et al., Nanoswitch-linked immunosorbent assay (NLISA) for fast, sensitive, and specific protein detection. PNAS. 114, 10367-10372 (2017)) and microRNA (See e.g., A. R. Chandrasekaran, et al., Cellular microRNA detection with miRacles: microRNA-activated conditional looping of engineered switches. Science Advances. 5, eaau9443 (2019)) detection.
The simple DNA nanoswitch-based assay for detection of viral RNA overcomes some limitations of currently available methods for clinical detection of RNA viruses in resource-limited areas. These include 1) robust detection without enzymes or equipment, 2) maintaining low-cost and simplicity, and 3) providing specificity and versatility. Surprisingly, the current COVID-19 pandemic has shown us that these problems can affect rich countries as well, with many struggling to have testing outpace viral spread.
The intrinsically high signal of the nanoswitches is enhanced here with a new “target multiplication” strategy where viral RNA fragmentation is used to multiply the number of targets, and thus increase the signal intensity. Using this approach, excellent levels of detection in urine were reached without the use of enzyme-mediated amplification strategies. This is of significance because enzymes can be key drivers of assay cost and complexity due to requirements including cold storage/transportation, special buffers and reagents, and strict operating temperatures. These factors make enzymatic assays difficult for field use or for use in low resource areas without modern lab infrastructure. Despite these challenges, most currently available techniques rely on enzyme triggered amplification. (See e.g., D. Musso, D. J. Gubler, Zika Virus. Clinical Microbiology Reviews. 29, 487-524 (2016); K. Pardee, et al., Rapid, Low-Cost Detection of Zika Virus Using Programmable Biomolecular Components. Cell. 165, 1255-1266 (2016); J. S. Gootenberg, et al., Multiplexed and portable nucleic acid detection platform with Cas13, Cas12a, and Csm6. Science. 360, 439-444 (2018)). For the assays of the present disclosure, compatibility and dramatic signal improvement have been demonstrated with an optional enzymatic pre-amplification step (
Two key features of the approach are simplicity and low cost. The DNA nanoswitches align with the goals of “frugal science” movement, where cost and accessibility to new technologies are valued alongside typical performance metrics (See e.g., G. Whitesides, The frugal way: The promise of cost-conscious science. The Economist: The World in 2012 (2011), p. 154; and S. Reardon, Frugal science gets DIY diagnostics to world's poorest. New Scientist. 219, 20-21 (2013)).
In embodiments, the nanoswitches cost around 1 penny per reaction and can be stored dry at room temperature for at least a month, and could be delivered globally without transportation or biosafety concerns. The assay consists of few steps and can be performed in a matter of hours with limited laboratory needs (
The emergence of a highly contagious novel coronavirus in 2019 led to an unprecedented need for large scale diagnostic testing. This increased testing also created problematic reagent bottlenecks for routinely used assays, necessitating the development of alternate diagnostic tests that are low-cost and sensitive with rapid turnaround time. Here, a rapid diagnostic test for SARS-CoV-2 is demonstrated eliminating the need for costly enzymes. Embodiments include one or more DNA nanoswitches that respond to segments of the viral RNA by a change in shape that is readable by gel electrophoresis. Embodiments include a new multi-targeting approach and improve the limit of detection, and a new workflow that integrates with bufferless gel cartridges provides results in <1 hour without laboratory equipment. Embodiments of the present disclosure are applied to a cohort of clinical samples, and have excellent specificity and sensitivity. Since the method embodiments directly detects viral RNA, it is less likely to report false positives and also directly shows the level of virus. The healthcare battle against the COVID-19 pandemic will benefit from the embodiments of the present disclosure, both for rapid onsite testing as well as for monitoring viral loads in recovering patients.
A novel coronavirus, SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2), has caused an outbreak of the disease COVID-19, (See e.g., Wang, C et al., A novel coronavirus outbreak of global health concern. The Lancet 395, 470-473 (2020)| and Zhu, N. et al. A Novel Coronavirus from Patients with Pneumonia in China, 2019. New England Journal of Medicine 382, 727-733 (2020)) with over 270 million reported cases and 5.3 million deaths globally since the start of the pandemic. (See e.g., WHO Coronavirus (COVID-19) Dashboard. https://covid19.who.int).
The pandemic highlights the need for development of rapid and low-cost alternate detection strategies for large scale testing. (See e.g., Udugama, B. et al. Diagnosing COVID-19: The Disease and Tools for Detection. ACS Nano 14, 3822-3835 (2020)). The gold-standard method for viral detection in clinical samples is RT-PCR (real-time polymerase chain reaction), which involves nucleic acid amplification and its monitoring via fluorescence. PCR-based tests, however, require days for diagnosing patients with suspected SARS-CoV-2, thus creating a gap in COVID positive individuals transmitting the virus before the results are obtained. Antigen tests can also be used to detect the spike protein on SARS-CoV-2 virions using lateral flow assay technology. These tests have been developed and marketed as rapid home-based tests, but are substantially less sensitive than PCR tests and still costly enough to limit their frequent use among much of the population. Serology tests are rapid and require minimal equipment, but these tests do not contribute to curtailing the spread of the disease as it can take several days to weeks following symptom onset for a patient to accumulate detectable levels of antibodies. (See e.g., Zhang, W. et al. Molecular and serological investigation of 2019-nCoV infected patients: implication of multiple shedding routes. Emerging Microbes & Infections 9, 386-389 (2020)). At various points in the pandemic, large scale global testing has strained supply chains and created bottlenecks of the reagents required for the PCR assay (See e.g., Esbin, M. N. et al. Overcoming the bottleneck to widespread testing: A rapid review of nucleic acid testing approaches for COVID-19 detection. RNA rna.076232.120 (2020) doi:10.1261/rna.076232.120), or general scarcity of rapid antigen tests. These limitations and bottlenecks in testing have negatively affected the ability to control the pandemic. Thus, the focus of new diagnostic assays has shifted from solely high sensitivity to also include aspects of cost, turnaround time, and operation outside a lab by non-specialists. (See e.g., Mina, M. J. et al., Rethinking Covid-19 Test Sensitivity—A Strategy for Containment. New England Journal of Medicine 0, null (2020)). These alternate testing strategies provide rapid results that aid in quick diagnosis and thus control of disease spread in contrast to highly sensitive tests with longer turnaround times. (See e.g., Toward COVID-19 Testing Any Time, Anywhere. The Scientist Magazine® www.the-scientist.com/news-opinion/toward-covid-19-testing-any-time-anywhere-67906; and Even imperfect Covid-19 tests can help control the pandemic. STAT www.statnews.com/2020/08/20/even-imperfect-covid-19-tests-can-help-control-the-pandemic/ (2020)). Over the past year, the frequency and speed of testing was found to be more practically relevant than test sensitivity for large scale COVID-19 testing. (See e.g., Larremore, D. B. et al. Test sensitivity is secondary to frequency and turnaround time for COVID-19 screening. Science Advances 7, eabd5393).
The challenges faced by traditional in vitro diagnostic techniques, and the need for more frequent testing, have accelerated the development of alternative COVID-19 testing strategies (See e.g., Udugama, B. et al. Diagnosing COVID-19: The Disease and Tools for Detection. ACS Nano 14, 3822-3835 (2020)). Several nanotechnology-based approaches have also been developed for detecting SARS-CoV-2, including those based on nanoparticles (See e.g., Moitra, P., et al., Selective Naked-Eye Detection of SARS-CoV-2 Mediated by N Gene Targeted Antisense Oligonucleotide Capped Plasmonic Nanoparticles. ACS Nano 14, 7617-7627 (2020), DNAzymes (See e.g., Sundah, N. R. et al. Catalytic amplification by transition-state molecular switches for direct and sensitive detection of SARS-CoV-2. Science Advances 7, eabe5940 (2021)), carbon nanotubes (See e.g., Pinals, R. L. et al. Rapid SARS-CoV-2 Spike Protein Detection by Carbon Nanotube-Based Near-Infrared Nanosensors. Nano Lett. 21, 2272-2280 (2021)), graphene (See e.g., Torrente-Rodriguez, R. M. et al. SARS-CoV-2 RapidPlex: A Graphene-Based Multiplexed Telemedicine Platform for Rapid and Low-Cost COVID-19 Diagnosis and Monitoring. Matter 3, 1981-1998 (2020)) and quantum dots (See e.g., Li, C. et al. Synthesis of polystyrene-based fluorescent quantum dots nanolabel and its performance in H5N1 virus and SARS-CoV-2 antibody sensing. Talanta 225, 122064 (2021)), with microfluidic (See e.g., Funari, R., Chu, K.-Y. & Shen, A. Q. Detection of antibodies against SARS-CoV-2 spike protein by gold nanospikes in an opto-microfluidic chip. Biosensors and Bioelectronics 169, 112578 (2020)), lateral flow (See e.g., Huang, C., Wen, T., Shi, F.-J., Zeng, X.-Y. & Jiao, Y.-J. Rapid Detection of IgM Antibodies against the SARS-CoV-2 Virus via Colloidal Gold Nanoparticle-Based Lateral-Flow Assay. ACS Omega 5, 12550-12556 (2020)), and optical (See e.g., Liu, T. et al. Quantification of antibody avidities and accurate detection of SARS-CoV-2 antibodies in serum and saliva on plasmonic substrates. Nature Biomedical Engineering 4, 1188-1196 (2020)), readout systems. Among new materials developed for biosensing, DNA-based nanostructures have been used to detect nucleic acids (See e.g., Chandrasekaran, A. R., Zavala, J. & Halvorsen, K. Programmable DNA Nanoswitches for Detection of Nucleic Acid Sequences. ACS Sens. 1, 120-123 (2016)), proteins (See e.g., Raveendran, M., Lee, A. J., Sharma, R., Wälti, C. & Actis, P. Rational design of DNA nanostructures for single molecule biosensing. Nature Communications 11, 4384 (2020), small molecules (See e.g., Jing, C. et al., An electrochemical aptasensor for ATP based on a configuration-switchable tetrahedral DNA nanostructure. Anal. Methods 12, 3285-3289 (2020), ions (See e.g., Narayanaswamy, N. et al. A pH-correctable, DNA-based fluorescent reporter for organellar calcium. Nature Methods 16, 95 (2019)), and changes in pH (See e.g., Idili, A., Vallée-Bélisle, A. & Ricci, F. Programmable pH-Triggered DNA Nanoswitches. J. Am. Chem. Soc. 136, 5836-5839 (2014)) or temperature. (See e.g., Gareau, D., Desrosiers, A. & Vallée-Bélisle, A. Programmable Quantitative DNA Nanothermometers. Nano Lett. 16, 3976-3981 (2016).
Recently, the precise spatial positioning and programmable control over shape of DNA nanostructures has been used to elucidate viral vaccine design principles (See e.g., Veneziano, R. et al. Role of nanoscale antigen organization on B-cell activation probed using DNA origami. Nature Nanotechnology 15, 716-723 (2020), and in viral diagnostics and therapeutics. (See e.g., Kwon, P. S. et al. Designer DNA architecture offers precise and multivalent spatial pattern-recognition for viral sensing and inhibition. Nature Chemistry 12, 26-35 (2020)). Here, a DNA nanotechnology based SARS-CoV-2 detection assay is provided based on programmable DNA nanoswitches. In embodiments, the DNA nanoswitch based assay is suitable for non-enzymatic, direct detection of SARS-CoV-2 by targeting multiple fragments of the SARS-CoV-2 genome. Assay embodiments have excellent sensitivity and specificity to discriminate single nucleotide variants, low cost (<$1) and has a gel-based readout that can be easily adapted for low-resource setting. Clinical utility is demonstrated herein by detecting the viral RNA in 10 clinically tested COVID-19 positive and 5 negative samples.
Table 14 below shows suitable target RNA strands for detecting SARS-CoV-2 in accordance with the present disclosure.
Table 15 below shows suitable left or first detector strands for certain SARS-CoV-2 target strands above.
Table 16 below shows suitable right or second detector strands for certain SARS-CoV-2 target strands above. It is noted that Tables 15 and 16 show paired detector strands when compared against each other in ascending order.
Results and Discussion
The DNA nanoswitch is constructed based on DNA origami principles. Typically, in DNA origami, a long single stranded DNA is folded into specific two- or three-dimensional shapes using short complementary strands. (See e.g., Rothemund, P. W. K. Folding DNA to create nanoscale shapes and patterns. Nature 440, 297-302 (2006)). Here, a simple linear duplex (the “off” state) was created using a 7249-nt long scaffold strand (commercially available M13 bacteriophage viral genome) tiled with short complementary backbone oligonucleotides. (See e.g., Chandrasekaran, A. R., Zavala, J. & Halvorsen, K. Programmable DNA Nanoswitches for Detection of Nucleic Acid Sequences. ACS Sens. 1, 120-123 (2016)). Two of the oligonucleotides contain single stranded extensions (detectors) that are complementary to parts of a target nucleic acid (
Here, direct, non-enzymatic detection of SARS-CoV-2 viral RNA is provided. As a first step, a nanoswitch was designed with detectors that respond to a target sequence in SARS-CoV-2 that has been used as a PCR primer in other diagnostic tests. Using a synthetic RNA target, the nanoswitches could detect the SARS-CoV-2 fragment (
In a typical viral detection assay, the target concentration (˜fM level) is generally lower than the nanoswitch concentration (˜200-500 pM level). To assess more realistic conditions, the kinetics were evaluated for reactions with excess nanoswitch (
More specifically,
Having satisfied the rapid testing requirement, the analytical sensitivity of the assay for viral targets was determined. Sensitivity for RNA targets has been reported in the 10-100 fM range. Using the process condition parameters of the present disclosure, the sensitivity of the assay was tested for the single target sequence, and determined a limit of detection (LOD) of 122 fM or 0.76 amol. To improve sensitivity, a new strategy for multi-targeting of different fragments of viral RNA was provided. The concept is based on the idea that fragmenting the viral RNA produces many discrete targets from a single viral RNA genome (
More specifically,
To design a multi-targeting nanoswitch, pairwise detectors were designed with a fixed distance apart with each pair offset along the length of the nanoswitch. Each pairwise detector detects a unique target to form a loop at a unique position on the nanoswitch but with a common loop size as other pairwise detectors. This causes individual loops to be indistinguishable when using gel electrophoresis as the readout. When the nanoswitch concentration>>target concentration (as with SARS-CoV-2 detection), this results in ability to detect multiple SARS-CoV-2 fragments with an additive signal (
Having proven the concept, the number of targets was expanded. Considering the long length of viral RNAs such as SARS-CoV-2 (˜30,000 nt) and the short length required for detection (˜20-60 nt), this idea can in principle be expanded to hundreds of targets and achieve a corresponding level of signal amplification. The 720 nt nanoswitch regions were dived into 30 nt segments to enable development of 24-target nanoswitches. Five such 24-target nanoswitches were provided to detect of up to 120 fragments that span the SARS-CoV-2 viral genome. The positive detection of each of the 120 individual targets was confirmed using DNA oligonucleotides (
In embodiments, the signal enhancement strategy includes robustly fragmenting the long viral RNA. To develop and test fragmentation protocols, an IVT RNA of the SARS-CoV-2 N-gene (1260 nt) was made and purified. Three fragmentation reagents were tested, a commercial buffer and two homemade buffers and settled on a homemade Tris-Magnesium reagent with 3 mM MgCl2. Using the homemade fragmentation buffer, the N-gene RNA was fragmented for various times from 1 minute to ˜1 hour. The products ran on a 2% agarose gel (
Next a nanoswitch design was obtained for the long IVT RNA. Long RNAs including viral RNAs are known to contain strong secondary structures, some of which may persist even as the RNA is fragmented into smaller pieces in the 200 nt range. Such structured regions could slow or otherwise impede binding to our DNA nanoswitch. Recent evidence in the lab suggested that longer detector regions enhanced capture of a 401 nt RNA. To see if that result applies to our fragmented viral RNA, nanoswitches were made and tested with detection regions of 15-30 nt such as 15, 20, 25 and 30 nt. It was found that overall detection signal clearly increased with longer detector lengths (
More specifically,
Choosing a short fragmentation time such as minutes, the IVT detection was expanded to a more realistic target RNA, spanning the full genome across 6 strands (Twist Biosciences). Using the process conditions of the present disclosure, the RNA with each of the 5 multi-targeting nanoswitches were detected in roughly equal proportion. Using a mixture of five 24-target nanoswitches that can detect 120 different SARS-CoV-2 fragments, an LOD for this full SARS-CoV-2 genome equivalent in aM is achieved.
To test our assay on clinical samples, 10 positive and 5 negative samples were obtained of RNA extracts from nasopharangeal swabs (previously confirmed by RT-qPCR). The assay was able to confirm all 5 negatives and registered positive in the samples of
More specifically,
Overall, assay embodiments, provide a unique non-enzymatic approach for direct detection of SARS-CoV-2 RNA. The method is shown to be sensitive enough to capture an excellent percentage of clinical positives in <1 hour of end-to-end assay time. The DNA nanoswitch approach has many advantages. The direct detection without enzymes eliminates high cost and complexity of enzymes including cold chain, and also provides a direct linear response that is proportional to viral load, which has been shown to be related to infectivity (See e.g., He, X. et al. Temporal dynamics in viral shedding and transmissibility of COVID-19. Nature Medicine 26, 672-675 (2020)) and severity of the disease (See e.g., Pujadas, E. et al. SARS-CoV-2 viral load predicts COVID-19 mortality. The Lancet Respiratory Medicine 8, e70 (2020)). A related advantage that became obvious during the pandemic is that assay embodiments of the present disclosure use different reagents than almost all other approaches, helping to diversify against supply shortages or bottlenecks.
In embodiments, the multi-targeting approach provides coverage over large portions of the genome, which can enable detection even while variants continue to evolve. A robust detection of the reference strain from Wuhan was shown as well as more recent Delta and Omicron variants. This can be seen as a particular advantage against PCR based methods, where several variants have caused problems in some standard diagnostic assays. Alternatively, the flexible design of the nanoswitch and the detectors does allow options for designing for specificity between variants, especially in those cases where there are numerous mutations. This same versatility also enables the method to be quickly adapted in response to new diseases.
As it stands, embodiments of the present disclosure fill a niche somewhere between diagnostic RT-qPCR and rapid antigen tests. In comparison with RT-qPCR, test embodiments of the present disclosure are likely to be substantially less expensive and faster turnaround, but with the downside of lower analytical sensitivity. Regarding sensitivity, it has been argued that preventing SARS-CoV-2 spread is more about frequent testing than sensitivity. (See e.g., Larremore, D. B. et al. Test sensitivity is secondary to frequency and turnaround time for COVID-19 screening. Science Advances 7, eabd5393). Compared to RT-qPCR, method embodiments, have another advantage of being more tolerant to minor contaminants and being operable without the intense cleanliness that is required of RT-qPCR. Some RT-qPCR has been noted to be affected by false positives due to sample contamination. (See e.g., Braunstein, G. D., Schwartz, L., Hymel, P. & Fielding, J. False Positive Results With SARS-CoV-2 RT-PCR Tests and How to Evaluate a RT-PCR-Positive Test for the Possibility of a False Positive Result. Journal of Occupational and Environmental Medicine 63, e159 (2021)). Compared to rapid antigen tests, the method embodiments provide similar time and equal or superior analytical sensitivity and likely similar or lower cost if developed into a commercial product.
This work demonstrates DNA nanoswitches with clinical samples and represents the first step in development for clinical use. Gel electrophoresis provides a low-cost and simple readout, but is commonly seen as messy and crude with a process not too dissimilar to making molded Jello. Cartridge-based bufferless gels may be a simple way to improve the process, and it has been demonstrated that the test can be ported with some loss of sensitivity to the Invitrogen E-gel system with a cell phone camera. It is believed that slight product variations of gel percentage and dye type could recover the lost sensitivity and make this a compelling solution. Other systems such as automated electrophoresis could be also adapted for this purpose, or possibly capillary electrophoresis.
Overall, embodiments of the present disclosure provide an entirely new way to detect SARS-CoV-2 RNA that is in a class of its own for direct detection of RNA without enzymes. The COVID-19 pandemic has illustrated how over-reliance on single types of tests can cause reagent shortages and workflow bottlenecks that can negatively impact disease control. (See e.g., Esbin, M. N. et al. Overcoming the bottleneck to widespread testing: A rapid review of nucleic acid testing approaches for COVID-19 detection. RNA rna.076232.120 (2020) doi:10.1261/rna.076232.120). The minimalist nature of embodiments of the present disclosure requires relatively few and low cost reagents, enabling the assay to be substantially lower than $1/test at scale. This type of test is adaptable for frequent home testing or surveillance testing. The programmability of the nanoswitches ensure that this method can be readily applied to other RNA viruses and perhaps make an impact on this or future disease outbreaks.
The entire disclosure of all applications, patents, and publications cited herein are herein incorporated by reference in their entirety. While the foregoing is directed to embodiments of the present disclosure, other and further embodiments of the disclosure may be devised without departing from the basic scope thereof.
The present disclosure claims priority or the benefit under 35 U.S.C. § 119 of U.S. provisional application No. 63/136,183 filed 11 Jan. 2021, which is herein entirely incorporated by reference.
This invention was made with governmental support under grant no. GM124720 awarded by the National Institutes of Health and grant no. CBET2030279 awarded by the National Science Foundation. The government has certain rights in this invention.
| Number | Date | Country | |
|---|---|---|---|
| 63136183 | Jan 2021 | US |
