Patent Application
20230120072
The development of this disclosure was funded in part by the Cancer Prevention and Research Institute of Texas (CPRIT) under Grant No. RP180147.
The present invention relates generally to the field of molecular biology. More particularly, it concerns methods for detecting and analyzing short single-stranded DNA, ultrashort single-stranded DNA and RNA in various biospecimens, and in particular in non-treated biospecimens.
Nucleic acid has emerged as an important analyte in molecular testing due to the richness of information in even minimal amount of material. Cellular genomic DNA or RNA is widely used in oncology, forensics, paternity testing, and research. Precision medicine relies on genomic information to provide guidance for individualized therapies, including diagnosis and prognosis for a variety of diseases including cancer, neurodegenerative diseases, and infectious diseases. The discovery of new classes of DNA biomarkers has preceded significant advances in diagnostics and benefited human health. The first wave of precision medicine was based on the analysis of germline mutations and SNPs from leukocyte or buccal swab samples to inform disease risk and drug dosage. Subsequently, nucleic acid biomarkers were expanded to include RNA expression patterns, DNA mutations in tumor tissue samples, circulating tumor cells (CTCs), cell-free DNA (cfDNA) and exosome-derived DNA from peripheral blood plasma. The classes, lengths, and sources of nucleic acid biomarkers are summarized in
One class of DNA biomarkers currently evaluated to have high translational value is cfDNA, double-stranded DNA in peripheral blood plasma with length around 165 base pairs (bp). Because cfDNA molecules are released through cell death or active secretion and are quickly cleared from the bloodstream with a half-life between 5 and 150 min, they capture a “snapshot” of dying cells throughput the whole body. Cell-free DNA have had transformative impact on both non-invasive prenatal testing (NIPT), organ transplant rejection monitoring, and cancer therapy selection and remission monitoring. Other examples of nucleic acid biomarkers being extensively studies are micro RNAs (miRNAs), long non-coding RNA, and exosome-derived DNA and RNA.
Despite their active footprint in both translational medicine and research, the current methods for purification of nucleic acids, including circulating DNA from plasma, systematically excludes the purification of other nucleic acid biomarkers. The most commonly used methods in commercial products based on silica-DNA interactions, based on columns or beads (e.g. QIAamp circulating nucleic acid kit (Qiagen), Cobas cfDNA sample preparation kit (Roche), or Apostle Minimax High Efficiency Cell-Free DNA Isolation Kit (Beckman Coulter)) systematically fail to extract DNA shorter than about 50 nt because those DNA molecules fail to bind to the columns or beads (
Provided herein are DNA extraction methods that are suitable for capturing single-stranded nucleic acid molecules, or nucleic acid molecules with partially single-stranded domains from un-treated biospecimens. The capture methods only involve mixing and incubating biospecimens with probes and hybrid capture buffers. In some embodiments, the captured molecules are analyzed by next generation sequencing with amendments of appropriate sequencing adapters. With the direct capture from biospecimen (DCB) approach, human red blood cells (RBCs) were found to be highly enriched in short single-stranded DNA (sssDNA), which is the opposite of expectation because RBCs have long been believed to be deprived of DNA due to the lack of nuclei in mature RBCs. On the other hand, sssDNA was found to be depleted in human plasma. Furthermore, sssDNAs were also found in biospecimens from non-human species. These findings indicate that the sssDNA might be a distinct DNA type in human and other species existing in cell membrane- or RBC membrane-bound format.
In one embodiment, provided herein are mixtures for direct capture from red blood cells comprising (1) isolated red blood cells that do not contain greater than 1 part in 1000 white blood cells and (2) an oligonucleotide capture probe with length between 5 nt and 100 nt (e.g., between 5 nt and 90 nt, between 5 nt and 80 nt, between 5 nt and 70 nt, between 5 nt and 60 nt, between 5 nt and 50 nt, between 10 and 100 nt, between 10 nt and 90 nt, between 10 nt and 80 nt, between 10 and 70 nt, between 10 and 60 nt, between 10 and 50 nt, or any range derivable therein) comprising (a) degenerate LNA nucleotides at between 2 and 50 loci (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 9, or 50 loci, or any range derivable therein) that do not allow polymerase extension or ligation and that do not include electrochemically active component and (b) an affinity tag modification at 3′, wherein the mixture does not comprise reverse transcriptase. In some aspects, the biospecimen includes but is not limited to red blood cells isolated from venous blood from human or non-human animals. In some aspects, the biospecimen includes but is not limited to red blood cells isolated from arterial blood from human or non-human animals. In some aspects, the red blood cell samples are not subjected to (1) storage at temperature above 4° C. for more than 48 hrs after sample collection; (2) heating above 45° C.; (3) enzymatic treatment (e.g. protease treatment); (4) harsh chemical treatment (e.g. lysis treatment); and/or (5) harsh physical treatment including but is not limited to shearing, electroporation, sonication. In some aspects, the affinity tag in capture probe includes but is not limited to (1) noncovalent affinity tags such as biotin, and (2) covalent affinity tags (reaction handle) such as azide, alkyne functional groups. In some aspects, the oligonucleotide of the capture probe comprises one or more non-natural degenerate bases with non-natural backbone modifications, such as locked nucleic acids. In some aspects, the oligonucleotide of the capture probe comprises one or more non-natural degenerate bases with universal affinity, such as inosine or 5-nitroindole. In some aspects, the hybrid capture buffer comprises (1) cation with concentration greater than 1 mM, (2) tween 20 with volume concentration between 0.01% and 1%, (3) Tris with concentration between 1 mM to 100 mM, (4) ethylenediaminetetraacetic acid (EDTA) with concentration between 1 mM to 100 mM, (5) sodium dodecyl sulfate (SDS) with volume concentration between 0.01% and 1%, and/or (6) tetramethylammonium chloride (TMAC) with concentration between 0 and 3 M.
In one embodiment, provided herein are methods for capturing sssDNA from red blood cells (RBCs), the methods comprising (1) isolating RBCs from freshly drawn blood; (2) mixing isolated RBCs with a capture probe comprising oligonucleotide with length between 5 nt and 100 nt (e.g., between 5 nt and 90 nt, between 5 nt and 80 nt, between 5 nt and 70 nt, between 5 nt and 60 nt, between 5 nt and 50 nt, between 10 and 100 nt, between 10 nt and 90 nt, between 10 nt and 80 nt, between 10 and 70 nt, between 10 and 60 nt, between 10 and 50 nt, or any range derivable therein) and an affinity tag and a buffer; (3) incubating the mixture from (2) at temperature between 0° C. and 45° C. (e.g., 0, 1, 2, 3, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, or 45° C., or any range derivable therein) for between 1 second and 1 day (e.g., 1 second, 30 seconds, 1 minute, 2 minutes, 5 minutes, 30 minutes, 1 hour, 2 hours, 6 hours, 12 hours, 18 hours, or 24 hours, or any range derivable therein) to allow for hybridization between sssDNA and capture probe; (4) collecting the capture probes using the affinity tag; and (5) washing the collected capture probes to remove unbound substances and collecting captured DNA in elution buffer.
In some aspects, freshly drawn blood is collected in anticoagulant coated tubes. In some aspects, methods for red blood cell isolation include but are not limited to density gradient centrifugation, fluorescence-activated cell sorting (FACS), and white blood cell depletion using immunomagnetic cell separation. In some aspects, the biospecimens are not subjected to (1) storage at temperature above 4° C. for more than 48 hrs after sample collection; (2) freeze-thaw for total blood samples; (3) heating above 45° C.; (4) enzymatic treatment (e.g. protease treatment); (5) chemical treatment (e.g. lysis treatment); and/or (6) harsh physical treatment including but is not limited to shearing, electroporation, sonication.
In some aspects, the affinity tag in capture probe includes but is not limited to (1) noncovalent affinity tags such as biotin, and (2) covalent affinity tags (reaction handle) such as azide or alkyne functional groups. In some aspects, the oligonucleotide of the capture probe comprises unmodified degenerate base stretch between 5 nt and 100 nt (e.g., between 5 nt and 90 nt, between 5 nt and 80 nt, between 5 nt and 70 nt, between 5 nt and 60 nt, between 5 nt and 50 nt, between 10 and 100 nt, between 10 nt and 90 nt, between 10 nt and 80 nt, between 10 and 70 nt, between 10 and 60 nt, between 10 and 50 nt, or any range derivable therein). In some aspects, the oligonucleotide of the capture probe comprises DNA oligonucleotide between 5 nt and 100 nt (e.g., between 5 nt and 90 nt, between 5 nt and 80 nt, between 5 nt and 70 nt, between 5 nt and 60 nt, between 5 nt and 50 nt, between 10 and 100 nt, between 10 nt and 90 nt, between 10 nt and 80 nt, between 10 and 70 nt, between 10 and 60 nt, between 10 and 50 nt, or any range derivable therein). In some aspects, the oligonucleotide of the capture probe comprises one or more non-natural degenerate bases with non-natural backbone modifications, such as locked nucleic acids. In some aspects, the oligonucleotide of the capture probe comprises one or more non-natural degenerate bases with universal affinity, such as inosine or 5-nitroindole. In some aspects, the concentration of the capture probe is between 50 pM and 5 μM (e.g., 50 pM, 100 pM, 500 pM, 1 nM, 50 nM, 100 nM, 500 nM, 1 μM, or 5 μM, or any range derivable therein).
In some aspects, the hybrid capture buffer comprises (1) cation with concentration greater than 1 mM; (2) tween 20 with volume concentration between 0.01% and 1%; (3) Tris with concentration between 1 mM to 100 mM; (4) ethylenediaminetetraacetic acid (EDTA) with concentration between 1 mM to 100 mM; (5) sodium dodecyl sulfate (SDS) with volume concentration between 0.01% and 1%; and/or (6) tetramethylammonium chloride (TMAC) with concentration between 0 and 3 M.
In some aspects, the method comprises RNase treatment to retain only one species of nucleic acids.
In some aspects, the method comprises using ligation and/or PCR approaches to append terminal sequences at 5′ and/or 3′ of single-stranded nucleic acid molecules. The appended terminal sequences can be adapter and index sequences for high-throughput sequencing. In some aspects, the method comprises amplifying the index-appended single-stranded molecules with index primers to increase concentration. In some aspects, the high-throughput sequencing is performed via sequencing-by-synthesis. In some aspects, the high-throughput sequencing is performed via sequence-specific current measurements in conjunction with nanopores.
In one embodiment, provided herein are methods for using sssDNA as disease prognostic biomarkers and treatment predictive biomarkers, based on mutation sequence variance in sssDNA. In some aspect, the sssDNAs are extracted and prepared for sequencing via methods described herein. In some aspects, sssDNAs can be prepared for methylation analysis, wherein extracted sssDNAs treated with bisulfite conversion reagents to transform all unmethylated cytosine to uracil prior to library preparation for high-throughput sequencing. In some aspects, sssDNAs can be prepared for methylation analysis, wherein extracted sssDNAs treated with oxidization reagents (e.g. TET2) and APOBEC to transform all unmethylated cytosine to uracil prior to library preparation for high-throughput sequencing. In some aspects, the lengths of sssDNAs are analyzed from high-throughput sequencing data, and if the sssDNAs are longer than sequencing read length, their lengths are inferred from aligned genomic positions of pair-end reads. In some aspects, genetic alterations, including but are not limited to single nucleotide variation, deletion, insertion, translocation and inversion, are analyzed to evaluate their association with disease and disease status. In some aspects, epigenetic alterations, most likely methylation patterns, are analyzed to evaluate their association with disease and disease status. In some aspects, expression profiles, including but are not limited to point mutations, fusion mutations, and expression levels, are analyzed to evaluate their association with disease and disease status.
In one embodiment, provided herein are methods for using sssDNA as disease prognostic biomarkers and treatment predictive biomarkers, based on quantitative relative concentration of sssDNA at different genome loci. In some aspects, the sssDNAs are extracted and prepared for sequencing via methods described herein. In some aspects, the lengths of sssDNAs are analyzed from high-throughput sequencing data, and if the sssDNAs are longer than sequencing read length, their lengths are inferred from aligned genomic positions of pair-end reads. In some aspects, the total concentrations of sssDNAs in biospecimens or in different compartment of biospecimens are estimated via spiking-in of synthetic reference sssDNA strands. In some aspects, sssDNAs aligned to different genomic loci are normalized to those aligned to reference loci (e.g., housekeeping genes, Alu sequences) to estimate relative concentrations at different genomic loci. In some aspects, the genomic loci of interest include but is not limited to promoter regions, 5′- and 3-′ UTRs, oncogenes, tumor suppressor genes, genes regulating immune responses or neurological activities. In some aspects, metagenomics of sssDNAs is analyzed for DNA concentrations of different bacteria populations. In some aspects, captured sssDNAs are analyzed for aneuploidy related to non-invasive prenatal testing (NIPT) or cancer copy number variation.
In one embodiment, provided herein are methods for the direct capture and extraction of single-stranded DNA (ssDNA) from a biospecimen, the methods comprising: (a) incubating a non-treated biospecimen with a DNA probe comprising an affinity tag and an oligonucleotide at a temperature between 0° C. and 45° C. (e.g., 0, 1, 2, 3, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, or 45° C., or any range derivable therein) in a solution comprising between 0.05 molar and 6 molar monovalent cations, or comprising between 0.001 molar and 2 molar divalent cations, or comprising both between 0.05 molar and 6 molar monovalent cations and between 0.001 molar and 2 molar divalent cations, for between 1 second and 1 day (e.g., 1 second, 30 seconds, 1 minute, 2 minutes, 5 minutes, 30 minutes, 1 hour, 2 hours, 6 hours, 12 hours, 18 hours, or 24 hours, or any range derivable therein) to allow for hybridization between the DNA probe and ssDNA in the biospecimen; (b) collecting the DNA probes using the affinity tag; and (c) washing the collected DNA probes to remove any non-hybridized contaminates from the biospecimen.
In one embodiment, provided herein are methods for direct capture and extraction of RNA from a biospecimen, the methods comprising: (a) incubating a non-treated biospecimen with an RNase inhibitor and a DNA probe comprising an affinity tag and an oligonucleotide at a temperature between 0° C. and 45° C. (e.g., 0, 1, 2, 3, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, or 45° C., or any range derivable therein)in a solution comprising between 0.05 molar and 6 molar monovalent cations, or comprising between 0.001 molar and 2 molar divalent cations, or comprising both between 0.05 molar and 6 molar monovalent cations and between 0.001 molar and 2 molar divalent cations, for between 1 second and 1 day (e.g., 1 second, 30 seconds, 1 minute, 2 minutes, 5 minutes, 30 minutes, 1 hour, 2 hours, 6 hours, 12 hours, 18 hours, or 24 hours, or any range derivable therein) to allow for hybridization between the DNA probe and RNA in the biospecimen; (b) collecting the DNA probes using the affinity tag; and (c) washing the collected DNA probes to remove any non-hybridized contaminates from the biospecimen.
In some aspects of any of the above embodiments, the non-treated biospecimen has not been heated above 45° C. prior to performing the method, has not undergone any biological treatments prior to performing the method, has not undergone any enzymatic reactions prior to performing the method, has not been treated with proteinase K prior to performing the method, has not undergone any chemical treatments prior to performing the method, has not undergone any harsh physical treatments prior to performing the method, has not been sheared prior to performing the method, has not been electroporated prior to performing the method, and/or has not been sonicated prior to performing the method.
In one embodiment, provided herein are methods for direct capture and extraction of single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA) from a biospecimen, the method comprising: (a) heating the biospecimen at a minimum of 90° C. for a minimum of 10 seconds to allow for denaturation of dsDNA; (b) contacting the biospecimen with a capture probe comprising an oligonucleotide having a length between 5 nt and 100 nt (e.g., between 5 nt and 90 nt, between 5 nt and 80 nt, between 5 nt and 70 nt, between 5 nt and 60 nt, between 5 nt and 50 nt, between 10 and 100 nt, between 10 nt and 90 nt, between 10 nt and 80 nt, between 10 and 70 nt, between 10 and 60 nt, between 10 and 50 nt, or any range derivable therein) and an affinity tag that allows for strong association with a solid-state substance; (c) incubating the biospecimen with the capture probe at a temperature between 0° C. and 45° C. (e.g., 0, 1, 2, 3, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, or 45° C., or any range derivable therein) for between 1 second and 1 day (e.g., 1 second, 30 seconds, 1 minute, 2 minutes, 5 minutes, 30 minutes, 1 hour, 2 hours, 6 hours, 12 hours, 18 hours, or 24 hours, or any range derivable therein) to allow for hybridization between the capture probe and nucleic acids in the biospecimen; (d) collecting the capture probes using the affinity tag; and (e) washing the collected capture probes to remove any non-hybridized contaminates from the biospecimen and collecting the capture nucleic acid.
In some aspects, the biospecimen comprises isolated red blood cells, isolated platelets, isolated white blood cells, blood, plasma, serum, urine, cerebrospinal fluid, and/or sputum. In some aspects of any of the above embodiments, the biospecimen is selected from the group consisting of plasma, serum, blood, urine, cerebrospinal fluid, and sputum. In some aspects, the biospecimen is from a human, an animal, a plant, or a bacterium. In some aspects, the biospecimen is a human biospecimen, and wherein the extracted ssDNA is human. In some aspects, the biospecimen is a human microbiome specimen. In some aspects, the human microbiome specimen is an oral, a skin, a vaginal, or a fecal biospecimen.
In some aspects, the biospecimen has not undergone any biological treatments prior to performing the method, has not undergone any enzymatic reactions prior to performing the method, has not been treated with proteinase K prior to performing the method, has not undergone any chemical treatments prior to performing the method, has not been lysed prior to performing the method, has not undergone any harsh physical treatments prior to performing the method, has not been sheared prior to performing the method, has not been electroporated prior to performing the method, and/or has not been sonicated prior to performing the method. In some aspects, the biospecimen is treated with a protease prior to step (a). In some aspects, the biospecimen has not been stored at a temperature above 4° C. for more than 48 hours prior to performing the method.
In some aspects of any of the above embodiments, the affinity tag is a noncovalent affinity tag, such as, for example biotin. In some aspects of any of the above embodiments, step (d) is performed via streptavidin-coated magnetic beads and collecting is performed using a magnet. In some aspects of any of the above embodiments, step (d) is performed via streptavidin-coated agarose beads and collecting is performed using centrifugal force. In some aspects of any of the above embodiments, the affinity tag is a covalent affinity tag (e.g., a reaction handle), such as, for example, an azide or alkyne functional group.
In some aspects of any of the above embodiments, the oligonucleotide of the capture probe comprises a region of degenerate bases. The region of degenerate bases may comprise between 5 and 100 degenerate bases (e.g., about 10 degenerate bases; e.g., between 5 and 90 degenerate bases, between 5 and 80 degenerate bases, between 5 and 70 degenerate bases, between 5 and 60 degenerate bases, between 5 and 50 degenerate bases, between 10 and 100 degenerate bases, between 10 and 90 degenerate bases, between 10 and 80 degenerate bases, between 10 and 70 degenerate bases, between 10 and 60 degenerate bases, between 10 and 50 degenerate bases, or any range derivable therein). Each degenerate base position may be any one of A, G, T or C. The region of degenerate bases may be located at the 5′ end of the oligonucleotide. In some aspects of any of the above embodiments, the oligonucleotide may further comprise a region of known bases. The region of known bases may comprise about 5 thymidines. The region of known bases may be located between the region of degenerate bases and the affinity tag.
In some aspects, the oligonucleotide of the capture probe is a DNA oligonucleotide. In some aspects, the oligonucleotide of the capture probe comprises one or more non-natural degenerate bases with non-natural backbone modifications. In some aspects, the oligonucleotide of the capture probe comprises locked nucleic acids. In some aspects, the oligonucleotide of the capture probe comprises one or more non-natural degenerate bases with universal affinity. In some aspects, the non-natural degenerate bases with universal affinity are inosine or 5-nitroindole. In some aspects, the concentration of the capture probe is between 50 pM and 5 μM (e.g., 50 pM, 100 pM, 500 pM, 1 nM, 50 nM, 100 nM, 500 nM, 1 μM, or 5 μM, or any range derivable therein).
In some aspects, step (b) further comprises contacting the biospecimen with a hybrid capture buffer, wherein the hybrid capture buffer comprises 100 mM to 1 M sodium chloride, 0.01% (v/v) to 1% (v/v) Tween20, 1 mM to 100 mM Tris, 1 mM to 100 mM ethylenediaminetetraacetic acid (EDTA), 0.01% (v/v) to 1% (v/v) doium dodecyl sulfate (SDS), and 0 M to 3 M tetramethylammonium chloride (TMAC). In some aspects, the hybrid capture buffer comprises between 0.05 molar and 6 molar monovalent cations, or between 0.001 molar and 2 molar divalent cations, or both between 0.05 molar and 6 molar monovalent cations and between 0.001 molar and 2 molar divalent cations.
In some aspects of any of the above embodiments, the capture probe in step (a) is not conjugated to a solid support. In certain aspects of any of the above embodiments, the methods are performed without an anion exchange medium.
In some aspects of any of the above embodiments, the hybridization in step (a) is direct hybridization between the capture probe and ssDNA or RNA in the biospecimen.
In some aspects, the methods comprise treating the biospecimen with an RNase.
In some aspects of any of the above embodiments, the methods further comprise eluting the hybridized nucleic acid from the capture probe. In some aspects of any of the above embodiments, the methods further comprise preparing an NGS library using the eluted nucleic acid. In some aspects, the methods further comprise using ligation and/or PCR to append terminal sequences on the 5′ and/or 3′ ends of the captured single-stranded nucleic acid molecules. In some aspects, the terminal sequences are adapter and index sequences for high-throughput sequencing. In some aspects, the methods further comprise amplifying the index-appended single-stranded molecules using index primers. In some aspects, during the process of NGS library preparation, the extracted nucleic acid is not amplified in a sequence-specific manner. In some aspects of any of the above embodiments, the methods further comprise performing high-throughput sequencing on the NGS library. In some aspects, the high-throughput sequencing is performed via sequencing-by-synthesis. In some aspects, the high-throughput sequencing is performed via sequence-specific current measurements in conjunction with nanopores. In some aspects of any of the above embodiments, the methods further comprise analyzing the sequences of the nucleic acid to predict disease in or select a treatment for a patient from whom the biospecimen was obtained. In some aspects of any of the above embodiments, the methods further comprise analyzing the relative concentrations of the ssDNA derived from various genomic loci to predict disease in or select a treatment for a patient from whom the biospecimen was obtained.
In some aspects of any of the above embodiments, the biospecimen is a human biospecimen, and the extracted nucleic acid is human. In some aspects of any of the above embodiments, the methods are methods of selectively isolating ssDNA or RNA.
As used herein, “essentially free,” in terms of a specified component, is used herein to mean that none of the specified component has been purposefully formulated into a composition and/or is present only as a contaminant or in trace amounts. The total amount of the specified component resulting from any unintended contamination of a composition is therefore well below 0.05%, preferably below 0.01%. Most preferred is a composition in which no amount of the specified component can be detected with standard analytical methods.
As used herein the specification, “a” or “an” may mean one or more. As used herein in the claim(s), when used in conjunction with the word “comprising,” the words “a” or “an” may mean one or more than one.
The use of the term “or” in the claims is used to mean “and/or” unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and “and/or.” As used herein “another” may mean at least a second or more.
Throughout this application, the term “about” is used to indicate that a value includes the inherent variation of error for the device, the method being employed to determine the value, the variation that exists among the study subjects, or a value that is within 10% of a stated value.
Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.
The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.
Hybrid capture-based methods to extract single-stranded DNA or RNA directly from non-treated biospecimens are provided herein. These methods allow for the discovery of unexplored short single-stranded DNA (sssDNA, mean length 50 nt) and ultrashort single-stranded DNA (ussDNA, mean length 15 nt) of human origin present in plasma. The DNA or RNA extracted using the disclosed methods here can be used as disease prognostic biomarkers and treatment predictive biomarkers. For example, the DNA or RNA extracted can be sequenced to identify mutation sequence variance or quantitative relative concentrations of single or multiple DNA or RNA molecules.
Compared to previous methods to extract DNA or RNA from biospecimen, the present methods can be directly applied to non-treated biospecimens, such as plasma, serum, blood, urine, cerebrospinal fluid, and sputum. In addition, the present methods are hybrid-capture based, and thus overcome the loss of short DNA and single-stranded DNA in existing DNA extraction methods, which are based on silica-DNA interactions using columns or beads. The methods also enable the discovery of unexplored short single-stranded DNA (sssDNA, mean length 50 nt) and ultrashort single-stranded DNA (ussDNA, mean length 15 nt) of human origin present in plasma.
“Amplification,” as used herein, refers to any in vitro process for increasing the number of copies of a nucleotide sequence or sequences. Nucleic acid amplification results in the incorporation of nucleotides into DNA or RNA. As used herein, one amplification reaction may consist of many rounds of DNA replication. For example, one PCR reaction may consist of 30-100 “cycles” of denaturation and replication.
“Biospecimen,” as used herein, includes, but is not limited to, plasma, serum, blood, urine, cerebrospinal fluid, tears, lymph fluid, peritoneal fluid, ascites fluid, umbilical cord blood, amniotic fluid, and sputum. In some embodiments, a biospecimen may not be subjected to various treatments, such as chemical modification and fragmenting treatments. Fragmenting treatments include mechanical, sonic, chemical, enzymatic, degradation over time, etc. Chemical modifications include bisulfite conversion and methylation/demethylation.
In certain aspects, the “capture probes” have a stretch of about 10 (e.g., 7, 8, 9, 10, 11, or 12) degenerate nucleotides. The term “degenerate” as used herein refers to a nucleotide or series of nucleotides wherein the identity can be selected from a variety of choices of nucleotides, as opposed to a defined sequence. The capture probe sequence may be NNNNNNNNNNTTTTT/3Bio/ (SEQ ID NO: 1), wherein N represents positions containing any one of multiple nucleotides. As such, the capture probe may have a 5′ degenerate region (e.g., 10 N residues) and a 3′ region having a known sequence (e.g., five T residues). A probe library with 10 variable positions, and 4 possible nucleotides at each position is comprised of 410=1,048,576 members. In a particular embodiment, the capture probe oligonucleotides are biotin-functionalized at the 3′ end, and streptavidin-functionalized magnetic beads are added to solution after the hybridization reaction between the biospecimen and the probes. Washing the magnetic bead suspension in the vicinity of a magnet removes unbound molecules.
The term “ligase” as used herein refers to an enzyme that is capable of joining the 3′ hydroxyl terminus of one nucleic acid molecule to a 5′ phosphate terminus of a second nucleic acid molecule to form a single molecule. The ligase may be a DNA ligase or RNA ligase.
“Polymerase chain reaction,” or “PCR,” means a reaction for the in vitro amplification of specific DNA sequences by the simultaneous primer extension of complementary strands of DNA. In other words, PCR is a reaction for making multiple copies or replicates of a target nucleic acid flanked by primer binding sites, such reaction comprising one or more repetitions of the following steps: (i) denaturing the target nucleic acid, (ii) annealing primers to the primer binding sites, and (iii) extending the primers by a nucleic acid polymerase in the presence of nucleoside triphosphates. Usually, the reaction is cycled through different temperatures optimized for each step in a thermal cycler instrument. Particular temperatures, durations at each step, and rates of change between steps depend on many factors well-known to those of ordinary skill in the art.
“Primer” means an oligonucleotide, either natural or synthetic, that is capable, upon forming a duplex with a polynucleotide template, of acting as a point of initiation of nucleic acid synthesis and being extended from its 3′ end along the template so that an extended duplex is formed. The sequence of nucleotides added during the extension process is determined by the sequence of the template polynucleotide. Usually primers are extended by a DNA polymerase. Primers are generally of a length compatible with its use in synthesis of primer extension products, and are usually are in the range of between 8 to 100 nucleotides in length, such as 10 to 75, 15 to 60, 15 to 40, 18 to 30, 20 to 40, 21 to 50, 22 to 45, 25 to 40, and so on, more typically in the range of between 18-40, 20-35, 21-30 nucleotides long, and any length between the stated ranges. Typical primers can be in the range of between 10-50 nucleotides long, such as 15-45, 18-40, 20-30, 21-25 and so on, and any length between the stated ranges. In some embodiments, the primers are usually not more than about 10, 12, 15, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, or 70 nucleotides in length.
As used herein, a nucleic acid “region” or “domain” is a consecutive stretch of nucleotides of any length.
The term “nucleic acid” or “polynucleotide” will generally refer to at least one molecule or strand of DNA, RNA, DNA-RNA chimera or a derivative or analog thereof, comprising at least one nucleobase, such as, for example, a naturally occurring purine or pyrimidine base found in DNA (e.g., adenine “A,” guanine “G,” thymine “T” and cytosine “C”) or RNA (e.g. A, G, uracil “U” and C). The term “nucleic acid” encompasses the terms “oligonucleotide” and “polynucleotide.” Note that although oligonucleotide and polynucleotide are distinct terms of art, there is no exact dividing line between them and they are used interchangeably herein. These definitions generally refer to at least one single-stranded molecule, but in specific embodiments will also encompass at least one additional strand that is partially, substantially, or fully complementary to at least one single-stranded molecule. Thus, a nucleic acid may encompass at least one double-stranded molecule. As used herein, a single stranded nucleic acid may be denoted by the prefix “ss,” and a double-stranded nucleic acid by the prefix “ds.” Notably, ssDNA is composed of nucleotides, while dsDNA is composed of base pairs, i.e., complementary nucleotide pairs. The nucleic acid molecule can be transformed from RNA into DNA and from DNA into RNA. For example, and without limitation, mRNA can be created into complementary DNA (cDNA) using reverse transcriptase and DNA can be created into RNA using RNA polymerase. A nucleic acid molecule can be of biological or synthetic origin.
Nucleic acid(s) that are “complementary” or “complement(s)” are those that are capable of base-pairing according to the standard Watson-Crick, Hoogsteen or reverse Hoogsteen binding complementarity rules. As used herein, the term “complementary” or “complement(s)” may refer to nucleic acid(s) that are substantially complementary, as may be assessed by the same nucleotide comparison set forth above. The term “substantially complementary” may refer to a nucleic acid comprising at least one sequence of consecutive nucleobases, or semiconsecutive nucleobases if one or more nucleobase moieties are not present in the molecule, are capable of hybridizing to at least one nucleic acid strand or duplex even if less than all nucleobases do not base pair with a counterpart nucleobase. In certain embodiments, a “substantially complementary” nucleic acid contains at least one sequence in which about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, to about 100%, and any range therein, of the nucleobase sequence is capable of base-pairing with at least one single or double-stranded nucleic acid molecule during hybridization. In certain embodiments, the term “substantially complementary” refers to at least one nucleic acid that may hybridize to at least one nucleic acid strand or duplex in stringent conditions. In certain embodiments, a “partially complementary” nucleic acid comprises at least one sequence that may hybridize in low stringency conditions to at least one single or double-stranded nucleic acid, or contains at least one sequence in which less than about 70% of the nucleobase sequence is capable of base-pairing with at least one single or double-stranded nucleic acid molecule during hybridization.
A “nucleoside” is a base-sugar combination, i.e., a nucleotide lacking a phosphate. It is recognized in the art that there is a certain inter-changeability in usage of the terms nucleoside and nucleotide. For example, the nucleotide deoxyuridine triphosphate, dUTP, is a deoxyribonucleoside triphosphate. After incorporation into DNA, it serves as a DNA monomer, formally being deoxyuridylate, i.e., dUMP or deoxyuridine monophosphate. One may say that one incorporates dUTP into DNA even though there is no dUTP moiety in the resultant DNA. Similarly, one may say that one incorporates deoxyuridine into DNA even though that is only a part of the substrate molecule.
“Nucleotide,” as used herein, is a term of art that refers to a base-sugar-phosphate combination. Nucleotides are the monomeric units of nucleic acid polymers, i.e., of DNA and RNA. The term includes ribonucleotide triphosphates, such as rATP, rCTP, rGTP, or rUTP, and deoxyribonucleotide triphosphates, such as dATP, dCTP, dUTP, dGTP, or dTTP.
“Solid support,” as used herein, means a solid carrier, including, but not limited to, a microtiter plate, beads (e.g., magnetic, glass, plastic, or metal coated beads), slides (e.g., glass or gold-coated slides), micro- or nano-particles, solid support platinum, palladium, microfluidization chamber, or channel carbon. In some cases, a solid support may be a solid support based on silicon oxide, a plastic polymer-based solid support (e.g., nylon, nitrocellulose or polyvinyl fluoride-based solid support), or a bio-based polymer (e.g., cross-linked dextran or cellulose-based solid support) solid support. A capture probe may be able to be pulled-down, directly or indirectly, using a solid support. For example, biotin can be a component of the capture probe, which can interact with a streptavidin-coated solid support.
In one embodiment, the direct capture approach is applied to extract single-stranded DNA from different blood components, namely, plasma, red blood cell layer, and white blood cell layer. Investigating the sssDNA content in the RBC layer, which is believed to be deprived of nucleic acids, is of particular interest.
RBC layer was separated from total blood by density gradient centrifugation. Freshly drawn blood was separated by centrifuging at 1,500×g for 20 min at room temperature. The upper clear plasma layer was first removed without interrupting the interface, and the interface was gently disrupted and moved to the side by a P1000 tip. The RBC was then collected by slowly drawing from the bottom-most liquid and leaving some RBC layer with the interface to avoid white blood cell contamination.
The isolated RBCs are mixed to capture probe and hybrid capture buffer and incubate at room temperature for 2 hrs with shaking to allow hybridization of sssDNA and capture probe. The capture probe is a 10-mer with degenerated LNA bases and biotin modification (5′-+N+N+N+N+N+N+N+N+N+N/iSp18//3Bio/-3′ (SEQ ID NO: 2)). The hybrid capture reaction comprises 2 μM of capture probe, 0.5 M NaCl, 1× TE, and 0.1% Tween-20.
Next, MyOne C1 streptavidin beads were added to the mixture, and incubated at room temperature for 30 min. The tube containing reaction mixture was put on a magnetic rack to remove and discard supernatant, and the remaining streptavidin beads were washed with buffer containing 0.5 M NaCl, 1× TE, and 0.1% Tween-20. Captured DNA was released from streptavidin beads by heating beads at 95° C. in water (
As described herein, methods for extracting short single-stranded DNA (ssDNA) using the direct capture from biospecimen (DCB) methods can be performed, for example, on human plasma samples. The DBC method can also be applied to biospecimens derived from non-human species, including plasma sample from monkey, plasma from mouse arterial blood, freshly prepared orange juice and peach juice, and milk. These methods provide for the detection and analysis of unexplored short single-stranded DNA (sssDNA, mean length 50 nt) and ultrashort single-stranded DNA (ussDNA, mean length 15 nt) of human origin present in plasma. The concentrations (in ng/mL) of sssDNA and ussDNA are higher than that of cfDNA (around 167 bp).
High-yield extraction of short single-stranded DNA can be achieved by the direct application of degenerate poly-N DNA probes to blood plasma to allow hybridization of short single-stranded DNA to the probes. The DCB workflow is summarized in
To co-extract cell-free DNA along with ssDNA, the plasma is first treated with protease and heat-denatured prior to DCB. Because the concentrations of cfDNA in the plasma are low, it is highly unlikely that denatured dsDNA rehybridizes on the timescale of the subsequent magnetic bead separation.
In one embodiment, heat-denatured plasma samples are prepared by first digesting proteins in the plasma using Protease K (56° C., 30 min), and then incubating at 98° C. for 15 min to denature the DNA and deactivate Protease K. In another embodiment, unprocessed plasma is directly used as input for DCB.
Unprocessed or heat-denatured plasma samples were then mixed with the capture probe, NaCl solution, TE buffer, and Tween-20 to result in a mixture containing 2 mM capture probe, 0.5 M NaCl, 0.8× TE, and 0.08% Tween-20. The capture probe sequence was NNNNNNNNNNTTTTT/3Bio/(SEQ ID NO: 1). The hybridization reaction was incubated at room temperature (25° C.) for 2 hrs. Next, MyOne C1 streptavidin beads were added to the mixture and incubated at room temperature for 30 min. The tube containing the reaction mixture was put on a magnetic rack to remove and discard supernatant, and the remaining streptavidin beads were washed with buffer containing 0.5 M NaCl, 1× TE, and 0.1% Tween-20. Captured DNA was released from streptavidin beads by heating beads at 95° C. in water (
In some embodiments, the captured sssDNAs are amended with Illumina sequencing adapters and sequenced on Miseq. The subsequent NGS library preparation process for sssDNA extracted from DCB or RBC utilizes the CircLigase enzyme, which acts on single-stranded DNA (
The library was sequenced by Miseq. After sequencing, NGS adapter sequences were first removed from paired-end NGS reads, and low-quality reads were also removed. Reads that are too short (length≤4 nt) were removed, because they are likely adapter dimers. Non-paired reads were also removed. Sequences with lengths between 5 nt and 150 nt needed to be perfectly paired, and sequences with lengths between 151 nt and 290 nt needed to have at least 10 paired bases in the middle of the sequence (
Different capture probes were tested to improve on-target rate and reduce artifact derived from residual capture probe included in the final library. Four capture probes were tested and fraction of capture probe-derived reads were summarized in Table 1. Comparing to TTTTT as the spacer between poly N and 3′ biotin, spacer that cannot be recognized by polymerase (such as iSp3 and iSp9 from IDT) reduced artifacts from probes. The probe-derived sequences were further removed by using Locked nucleic acid (LNA) probe with a spacer that cannot be recognized by polymerase.
The technology herein includes kits for performing the direct capture from biospecimen methods provided herein. A “kit” refers to a combination of physical elements. For example, a kit may include, for example, one or more components, such as randomer capture probes, as well as, streptavidin-coated beads, enzymes, reaction buffers, primers for NGS library preparation, an instruction sheet, and other elements useful to practice the technology described herein. These physical elements can be arranged in any way suitable for carrying out the disclosure.
The components of the kits may be packaged either in aqueous media or in lyophilized form. The container means of the kits will generally include at least one vial, test tube, flask, bottle, syringe or other container means, into which a component may be placed, and preferably, suitably aliquoted (e.g., aliquoted into the wells of a microtiter plate). Where there is more than one component in the kit, the kit also will generally contain a second, third or other additional container into which the additional components may be separately placed. However, various combinations of components may be comprised in a single vial. The kits of the present disclosure also will typically include a means for containing the nucleic acids, and any other reagent containers in close confinement for commercial sale. Such containers may include injection or blow molded plastic containers into which the desired vials are retained.
A kit will also include instructions for employing the kit components as well the use of any other reagent not included in the kit. Instructions may include variations that can be implemented. It is contemplated that such reagents are embodiments of kits of the disclosure. Such kits, however, are not limited to the particular items identified above.
The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.
Total blood was separated as previously described, from which RBC layer, WBC layer (with some RBCs) from the same healthy individual were prepared for sssDNA capture and NGS library preparation. The sequencing results were analyzed for length distribution and whole genome alignment.
Next, these reads were aligned using Bowtie 2 to the human genome, and over 90% of the reads mapped to the human genome (
Spike-in reference DNAs were used to estimate the concentration of sssDNA in plasma from a healthy volunteer. Reference DNAs were synthetic single-stranded DNAs with length of 20 nt, 30 nt, 40 nt, 50 nt, 60 nt and 70 nt, and at each length four different sequences were added to hybrid capture solution at 1 pM per strand. The capture mixture comprised 100 μL of human plasma, and 24 pM of total spike-in DNA in a total of 240 μL mixture.
(51 pM/100 μL plasma)×(240 μL)×(average size 35 nt)×(330 g/mol/nt)=1.4 ng/mL plasma.
Whether the DCB method primarily captures ssDNA was tested. Two spike-in ssDNA strands were added at 1 pM to hybrid capture solution, and the NGS reads aligned to their sequences are within 2-fold. However, when spike-in ssDNA2 was pre-annealed to its complementary strand and added to the system as dsDNA, its reads became less than 1% of the other spike-in ssDNA (
The DBC method was also applied to biospecimens derived from non-human species, including plasma sample from monkey, plasma from mouse arterial blood, freshly prepared orange juice and peach juice, and milk. Direct capture found similar distribution of short sssDNAs as seen in human specimens, and the captured sssDNAs displayed uniform distribution throughout whole genome of the corresponding species (
Direct capture from biospecimen (DCB) method for extracting ssDNA has been developed and tested in both non-treated and heat-denatured healthy plasma samples. The extracted ssDNA was analyzed by single-stranded sequencing library preparation and sequencing as described above. Two classes of unexplored single-stranded DNA have been discovered in human plasma.
The typical NGS results for one individual's plasma (both non-treated and heat-denatured) are summarized in
The reads were aligned to the human genome using Bowtie 2, and over 90% of the reads mapped to the human genome (
The concentration of ussDNA appears to be far higher than that of sssDNA based on the NGS sequencing results. Due to the short length of ussDNA (˜15 nt), the sequences map nonspecifically to the genomes of many different species, so it is difficult to verify if any given ussDNA molecule has a human origin. However, there is a high diversity of different ussDNA sequences, so it is likely that many ussDNA are of human origin.
The concentration of sssDNA was quantified through comparison to cfDNA by applying DCB after heat-denaturation of plasma (
All of the methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.
The following references, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference.
The present application claims the priority benefit of U.S. provisional application No. 62/951,069, filed Dec. 20, 2019, the entire contents of which is incorporated herein by reference.
This invention was made with government support under Grant No. R01 HG008752 awarded by the National Institutes of Health. The government has certain rights in the invention.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US20/66152 | 12/18/2020 | WO |
