Patent Application
20240368655
The instant application contains twenty-six (26) sequences which have been submitted in accordance with the ST.26 XML WIPO standard. Said XML file containing the sequences is entitled BCU0020US2-SEQ.xml and is approximately 27.6 kB in size, the contents of which are hereby incorporated in their entirety by reference.
The functions of natural nucleic acids such as DNA and RNA have transcended genetic information carriers and now encompass affinity reagents, molecular catalysts, nanostructures, data storage, and many others. However, the vulnerability of natural nucleic acids to nuclease-mediated degradation and the lack of chemical functionality have imposed a significant constraint on their applications.
Xeno(biotic) nucleic acids (XNAs) comprising a backbone other than the naturally occurring sugar-phosphate backbone present in DNA or RNA have been developed to minimize nuclease susceptibility. XNAs include bridged nucleic acids (BNAs), cyclohexene nucleic acids (CeNAs), 2′-deoxy-2′-fluoroarabino nucleic acids (FANAs), glycol nucleic acids (GNAs), 1,5-anhydrohexitol nucleic acids (HNAs), locked nucleic acids (LNAs), 2′-O-methyl ribonucleotides, morpholino nucleic acids (MNAs), peptide nucleic acids (PNAs), and threose nucleic acids (TNAs). The majority of XNA structures developed to date incorporate the same nucleobases adenine (A), cytosine (C), guanine (G), and thymine (T) or uracil (U) as the natural nucleic acids. To enable efficient uptake of XNA building blocks in enzymatic polymerization reactions, rational mutagenesis and directed evolution have generated engineered polymerases that have altered substrate specificity and can tolerate unnatural XNA nucleotide triphosphates (NTPs).
Installation of functional groups to the nucleobases has expanded the chemical repertoire and functions of nucleic acids. A variety of hydrophobic groups have been incorporated into DNA and RNA aptamers, including Slow Off-rate Modified Aptamers (SOMAmers) and highly functionalized nucleic acid polymers (HFNAPs). While this approach provides quantitative incorporation of base-modified nucleotides, bulky modifications often become challenging to incorporate and necessitate re-engineering of the polymerases.
An alternative strategy to introduce nucleobase modifications is incorporation of nucleotides comprising click handle-modified bases followed by efficient conjugation of a variety of chemical motifs via click chemistry. The click approach has introduced hydrophobic groups and carbohydrates onto DNA aptamers.
Described herein is the synthesis of a “click handle”-modified FANA (cmFANA) triphosphate nucleotides and nucleic acids comprising the same, e.g., 5-octa-1,7-diynyluracil 2′-deoxy-2′-fluoroarabino triphosphate (“C8-alkyne-FANA UTP”, compound 11 in
A better understanding of features and advantages of the present disclosure will be obtained by reference to the following detailed description, which sets forth illustrative embodiments of the disclosure, and the accompanying drawings.
While various embodiments of the present disclosure are described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous modifications and changes to, and variations and substitutions of, the embodiments described herein will be apparent to those skilled in the art without departing from the disclosure. It is understood that various alternatives to the embodiments described herein may be employed in practicing the disclosure. It is also understood that every embodiment of the disclosure may optionally be combined with any one or more of the other embodiments described herein which are consistent with that embodiment.
Where elements are presented in list format (e.g., in a Markush group), it is understood that each possible subgroup of the elements is also disclosed, and any one or more elements can be removed from the list or group.
It is also understood that, unless clearly indicated to the contrary, in any method described or claimed herein that includes more than one act or step, the order of the acts or steps of the method is not necessarily limited to the order in which the acts or steps of the method are recited, but the disclosure encompasses embodiments in which the order is so limited.
It is further understood that, in general, where an embodiment in the description or the claims is referred to as comprising one or more features, the disclosure also encompasses embodiments that consist of, or consist essentially of, such feature(s).
It is also understood that any embodiment of the disclosure, e.g., any embodiment found within the prior art, can be explicitly excluded from the claims, regardless of whether or not the specific exclusion is recited in the specification.
It is further understood that the present disclosure encompasses analogs, derivatives, salts, solvates, hydrates, clathrates and polymorphs of all of the compounds/substances disclosed herein, as appropriate. The specific recitation of “analogs”, “derivatives”, “salts”, “solvates”, “hydrates”, “clathrates” or “polymorphs” with respect to a compound/substance or a group of compounds/substances in certain instances of the disclosure shall not be interpreted as an intended omission of any of these forms in other instances of the disclosure where the compound/substance or the group of compounds/substances is mentioned without recitation of any of these forms.
It is also understood that the present disclosure encompasses all possible stereoisomers, including all possible diastereomers and enantiomers and racemic mixtures of enantiomers, of the compounds described herein, and not only the specific stereoisomers as indicated by drawn structure or nomenclature. Some embodiments of the disclosure relate to the specific stereoisomers indicated by drawn structure or nomenclature. The specific recitation of the phrase “or stereoisomers thereof” or the like with respect to a compound in certain instances of the disclosure shall not be interpreted as an intended omission of any of the other possible stereoisomers of the compound in other instances of the disclosure where the term “compound” is used without recitation of the phrase “or stereoisomers thereof” or the like.
Headings are included herein for reference and to aid in locating certain sections. Headings are not intended to limit the scope of the embodiments and concepts described in the sections under those headings, and those embodiments and concepts may have applicability in other sections throughout the entire disclosure.
All patent literature and all non-patent literature cited herein are incorporated herein by reference in their entirety to the same extent as if each patent literature or non-patent literature were specifically and individually indicated to be incorporated herein by reference in its entirety.
Unless defined otherwise or clearly indicated otherwise by their use herein, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which this application belongs.
As used in the specification and the appended claims, the indefinite articles “a” and “an” and the definite article “the” can include plural referents as well as singular referents unless specifically stated otherwise or the context clearly indicates otherwise.
The term “exemplary” as used herein means “serving as an example, instance or illustration”. Any embodiment or feature characterized herein as “exemplary” is not necessarily to be construed as preferred or advantageous over other embodiments or features.
The term “about” or “approximately” means an acceptable error for a particular value as determined by one of ordinary skill in the art, which depends in part on how the value is measured or determined. In certain embodiments, the term “about” or “approximately” means within one standard deviation. In some embodiments, when no particular margin of error (e.g., a standard deviation to a mean value given in a chart or table of data) is recited, the term “about” or “approximately” means that range which would encompass the recited value and the range which would be included by rounding up or down to the recited value as well, taking into account significant figures. In certain embodiments, the term “about” or “approximately” means within 10% or 5% of the specified value. Whenever the term “about” or “approximately” precedes the first numerical value in a series of two or more numerical values or in a series of two or more ranges of numerical values, the term “about” or “approximately” applies to each one of the numerical values in that series of numerical values or in that series of ranges of numerical values.
Whenever the term “at least” or “greater than” precedes the first numerical value in a series of two or more numerical values, the term “at least” or “greater than” applies to each one of the numerical values in that series of numerical values.
Whenever the term “no more than” or “less than” precedes the first numerical value in a series of two or more numerical values, the term “no more than” or “less than” applies to each one of the numerical values in that series of numerical values.
The term “compound” encompasses salts, solvates, hydrates, clathrates and polymorphs of that compound. A “solvate” of a compound comprises a stoichiometric or non-stoichiometric amount of a solvent (e.g., water, acetone or an alcohol [e.g., ethanol]) bound non-covalently to the compound. A “hydrate” of a compound comprises a stoichiometric or non-stoichiometric amount of water bound non-covalently to the compound. A “clathrate” of a compound contains molecules of a substance (e.g., a solvent) enclosed in a crystal structure of the compound. A “polymorph” of a compound is a crystalline form of the compound. The specific recitation of “salt”, “solvate”, “hydrate”, “clathrate” or “polymorph” with respect to a compound in certain instances of the disclosure shall not be interpreted as an intended omission of any of these forms in other instances of the disclosure where the term “compound” is used without recitation of any of these forms.
The term “polynucleotide” refers to a polymer composed of nucleotide units. Polynucleotides can contain naturally occurring nucleic acids (e.g., deoxyribonucleic acid [“DNA” ] and ribonucleic acid [“RNA” ]), or/and nucleic acid analogs.
“Aptamers” are short, single-stranded DNA or RNA (ssDNA or ssRNA) molecules that can selectively bind to a specific target, including proteins, peptides, carbohydrates, small molecules, toxins, and even live cells. The term stems from the Latin terms “aptus,” meaning to fit, and “meros,” meaning part. Aptamers assume a variety of shapes due to their tendency to form helices and single-stranded loops. They are extremely versatile and bind targets with high selectivity and specificity. Rather than primary sequence, aptamer binding is determined by formation of tertiary structure. Target recognition and binding involve three-dimensional, shape-dependent interactions as well as hydrophobic interactions, base-stacking, and intercalation. Aptamers bind because they “fit” their targets.
Although aptamers recognize and bind targets of interest just like antibodies, they have several advantages, including: reproducible high affinity & specificity; no or low batch-to-batch variation; increased thermal stability; non-immunogenic; and, lower costs of manufacturing. Canonical nucleic acid (i.e., DNA and RNA)-based aptamers suffer from two significant shortcomings: they are extremely susceptible to nuclease-mediated degradation; and, they lack chemical functionalities often critical to act as highly specific ligands for protein targets.
Aptamers may be created in a fashion in which they may contain one or more nucleic acid or nucleotide analogs. Nucleic acid or nucleotide analogs include without limitation those which have a non-naturally occurring base/nucleobase, have a sugar or non-sugar moiety other than 2′-deoxyribose or ribose, or engage in linkages with additional nucleotides other than the naturally occurring phosphodiester bond, or a combination thereof. Non-limiting examples of nucleic acid or nucleotide analogs include xeno(biotic) nucleic acids (XNAs) having a backbone other than the naturally occurring sugar-phosphate backbone present in DNA or RNA (e.g., bridged nucleic acids [BNAs], cyclohexene nucleic acids [CeNAs], 2′-deoxy-2′-fluoroarabino nucleic acids [FANAs], glycol nucleic acids [GNAs], 1,5-anhydrohexitol nucleic acids [1-NAs], locked nucleic acids [LNAs], 2′-O-methyl ribonucleotides, morpholino nucleic acids [MNAs], peptide nucleic acids [PNAs], and threose nucleic acids [TNAs]), phosphorothioates, phosphorodithioates, phosphorotriesters, phosphoramidates, boranophosphates, methylphosphonates, chiral-methyl phosphonates, and the like. DNA and RNA polynucleotides can be synthesized using a DNA or RNA polymerase or an automated DNA or RNA synthesizer. Polynucleotides containing nucleic acid analogs can be synthesized using, e.g., an engineered DNA or RNA polymerase that recognizes the nucleic acid analogs, a phosphoramidite strategy, or an automated peptide synthesizer in the case of PNAs. The term “nucleic acid molecule” typically refers to a larger polynucleotide. The term “oligonucleotide” typically refers to a shorter polynucleotide. In certain embodiments, an oligonucleotide contains no more than about 50 nucleotides. In some embodiments, when a polynucleotide sequence is represented by a DNA sequence (i.e., A, T, G, C), this also includes the corresponding, or the complementary, RNA sequence (i.e., A, U, G, C) in which “U” replaces “T”.
Conventional notation is used herein to describe polynucleotide sequences: the left-hand end of a single-stranded polynucleotide sequence is the 5′ end; the left-hand direction of a double-stranded polynucleotide sequence is referred to as the 5′ direction. The direction of 5′ to 3′ addition of nucleotides to a newly forming (daughter) DNA strand, or to a nascent RNA transcript, is referred to as the direction of replication or transcription, respectively. The DNA strand having a complementary, antiparallel sequence as a daughter DNA strand or a primary RNA transcript (the “coding strand”) is called the “template strand”. “Upstream” is toward the 5′ end of an RNA molecule, and “downstream” is toward its 3′ end. When considering double-stranded DNA, “upstream” is toward the 5′ end of the coding strand for the gene in question and “downstream” is toward the 3′ end of the coding strand, so the 3′ end of the template strand is upstream of the gene in question and the 5′ end of the template strand is downstream of the gene.
The term “primer” refers to a polynucleotide that is capable of specifically hybridizing to a designated polynucleotide template and providing a point of initiation for synthesis of a complementary polynucleotide. Such synthesis occurs when the polynucleotide primer is placed under conditions in which synthesis is induced, i.e., in the presence of nucleotides, a complementary polynucleotide template, and an agent for polymerization such as a DNA or RNA polymerase. A primer is typically single-stranded, but may be double-stranded. Primers are typically deoxyribonucleic acids, but a wide variety of synthetic and naturally occurring primers are useful for many applications. A primer is complementary to the template to which it is designed to hybridize and serves as a site for the initiation of synthesis, but need not reflect the exact sequence of the template. In such a case, specific hybridization of the primer to the template depends on the stringency of the hybridization conditions. A primer can be labeled with an agent that promotes isolation (e.g., biotin), separation (e.g., biotin) or detection (e.g., a fluorescent, chromogenic or radioactive moiety) of the coding strand in single-stranded form or the coding strand hybridized to the template strand.
To expand the chemical repertoire of XNA systems and incorporate a wide range of functional side chains, the disclosure describes a click handle-modified FANA (cmFANA) strategy employing 5-octa-1,7-diynyl FANA uracil triphosphate (“C8-alkyne-FANA UTP”, compound 11 in
The following embodiments of the present disclosure are provided by way of illustration and example:
A nucleotide compound designated 5-octa-1,7-diynyluracil 2′-deoxy-2′-fluoroarabino or nucleic acid comprising the same, wherein the nucleotide compound is of the following structure, or a salt, solvate or hydrate thereof:
A method of synthesizing 5-octa-1,7-diynyluracil 2′-deoxy-2′-fluoroarabino nucleic acid, or a salt, solvate or hydrate thereof, substantially as described in
A compound designated 5-octa-1,7-diynyluracil 2′-deoxy-2′-fluoroarabino nucleic acid triphosphate and having the following structure, or a salt, solvate or hydrate thereof:
A method of synthesizing 5-octa-1,7-diynyluracil 2′-deoxy-2′-fluoroarabino nucleic acid triphosphate, or a salt, solvate or hydrate thereof, substantially as described in
A method of synthesizing an aptamer, comprising mixing a template (e.g., a DNA template), a primer (e.g., a DNA primer), 5-octa-1,7-diynyluracil 2′-deoxy-2′-fluoroarabino nucleic acid triphosphate, and a wild-type or engineered polymerase (e.g., a wild-type or engineered DNA polymerase such as Thermococcus gorgonarius [Tgo] DNA polymerase).
The method of synthesizing an aptamer according to embodiment 5, further comprising mixing 2′-deoxy-2′-fluoroarabino nucleic acid (FANA) analogs of adenosine-5′-triphosphate (ATP), cytidine-5′-triphosphate (CTP) and guanosine-5′-triphosphate (GTP).
The method of synthesizing an aptamer according to embodiment 5 or 6, wherein the primer is labeled with an agent that promotes isolation (e.g., biotin), separation (e.g., biotin) or detection (e.g., a fluorescent or chromogenic compound) of a single-stranded or double-stranded aptamer comprising one or more 5-octa-1,7-diynyluracil 2′-deoxy-2′-fluoroarabino nucleotides.
A method of conjugating a single-stranded or double-stranded aptamer comprising one or more 5-octa-1,7-diynyluracil 2′-deoxy-2′-fluoroarabino nucleotides to an azide-functionalized compound (e.g., a carbohydrate) via a copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) reaction.
The following examples are intended only to illustrate the disclosure. Other procedures, methodologies, techniques, reagents and conditions may alternatively be used as appropriate.
Abbreviations: ACN=acetonitrile; AcOH=acetic acid; Bz=benzoyl; d=day(s); DAST=diethylaminosulfur trifluo ide (Et2NSF3); DCM=dichloromethane; DMF=N,N-dimethylformamide; mCPBA=meta-chloroperoxybenzoic acid; r.t.=room temperature; TBAF=tetra-n-butylammonium fluoride; TBDPS=tert-butyldiphenylsilyl; THF=tetrahydrofuran.
In the synthetic scheme shown in
Triphosphorylation of compound 6 was initially attempted using POCl3 and pyrophosphate following the conventional Yoshikawa phosphorylation protocol (
It was hypothesized that Thermococcus gorgonarius (Tgo) DNA polymerase could tolerate the 5-1,7-octadiyne modification on uracil and that the enzyme could incorporate FANA nucleotides. To investigate the hypothesis, a primer extension experiment was performed, in which a DNA primer-template complex was enzymatically extended by Tgo DNA polymerase using commercially available FANA nucleotide triphosphates (A, C, and G) as well as C8-alkyne-FANA UTP 11. The DNA template was prepared to contain a 40-nucleotide (nt) random region to avoid sequence bias, as well as a 24-nt poly-AAC tail at the 3′-end, which would not be transcribed, to provide a mobility difference between extension product (76 nt) and DNA template (100 nt) in denaturing polyacrylamide gel electrophoresis (PAGE). The DNA primer was labeled by a 5′-Cy5 fluorophore to allow easy detection of the extended product. A control experiment in which all FANA nucleotide triphosphates comprising natural bases (A, C, G and T) was also conducted to determine the impact of the 5-1,7-octadiyne modification of uracil on the primer extension reactivity. Tgo DNA polymerase was added in various amounts to confirm that the generation of the extended products was mediated by the Tgo DNA polymerase in a dose-dependent fashion. Both the primer extension reaction involving FANA NTPs of all natural bases and the one involving the FANA ATP, CTP, GTP, and compound 11 produced the full-length products in a polymerase dose-dependent fashion (
To unambiguously confirm the primer extension product, a cmFANA sequence was synthesized using a DNA template (T-ConA-XL, 98 nt including an 18-nt non-transcribed AAC repeating sequence at the 3′-end) encoding an aptamer targeting Concanavalin A (ConA), and a 5′-biotinylated DNA primer. After the primer extension reaction, the DNA-cmFANA heteroduplex was captured by streptavidin-coated magnetic particles, and the cmFANA polymer was separated from the DNA template strand by treating the captured heteroduplex with sodium hydroxide. The cmFANA polymer was then eluted from the magnetic particles by incubation with concentrated ammonium hydroxide for two hours. Electrospray ionization mass spectrometry (ESI-MS) analysis of the strand-separated cmFANA polymer suggested that the observed molecular weight of the cmFANA polymer (27,632.5 Da) was consistent with the expected value (27,632.5 Da). Taken together, these results indicated that the 5-1,7-octadiyne modification of uracil was well-tolerated by Tgo DNA polymerase and that C8-alkyne-FANA UTP could be quantitatively incorporated in a DNA-templated primer extension reaction mediated by this enzyme.
To demonstrate cmFANA polymer as a functionalizable synthetic genetic polymer for in vivo applications, its stability in human serum, which contains a variety of nucleases and mimics the typical environment for in vivo applications, was evaluated. To allow facile quantitation of the cmFANA polymer after human serum treatment, azido-fluorescein was conjugated to the cmFANA polymer via CuAAC (
Carbohydrate-modified nucleic acids have emerged as a promising technology to develop affinity reagents for glycan-binding proteins (GBPs) and vaccine epitopes. To demonstrate that cmFANA could serve as a general platform for carbohydrate-modified XNAs, click conjugation of azido-functionalized glycans with cmFANA polymer was evaluated (
Click conjugation of a disaccharide lactose and a trisaccharide 2,6-sialyllactose to cmFANA1 was investigated next. Lactose and commercially available 2,6-sialyllactose were equipped with an azido group at the reducing end by reaction with 2-chloro-1,3-dimethylimidazolinium chloride (DMC) and sodium azide in water (
Directed evolution of modified nucleic acids has become a powerful approach to discover affinity reagents and catalysts with enhanced functions. A critical step in the laboratory evolution process is amplification of the genetic information carried by the evolving population. Amplification is often challenging with base-modified nucleic acids due to an inability to be reverse-transcribed back into native DNA. The bulky carbohydrate-conjugated cmFANA structure raises the question of whether it could be directly reverse-transcribed back to DNA. A series of polymerases, including Taq, Bst LF*, Bst2.0, Bst3.0, Kod, Deep Vent, Tgo and Q5, was tested for their reverse transcriptase activity with cmFANA1 and Man-N3-conjugated cmFANA1. None of the polymerases tested could reverse-transcribe cmFANA1 or Man-N3-conjugated cmFANA1 (data not shown).
To circumvent the challenge of reverse transcription, an alternative strategy for amplifying the genetic information of a carbohydrate-conjugated cmFANA polymer based on DNA-display was devised. The DNA-display strategy was based on that developed by Temme and Krauss, Curr. Protoc. Chem. Biol., 7:73-92 (2015) in their selection with modified aptamers (SELMA) method, which involves using a hairpin structure to establish the critical phenotype-genotype linkage (
In
The feasibility of carbohydrate-conjugated cmFANA polymers to be evolved by in vitro selection was validated by a mock selection experiment (
The first portion of this disclosure described the development of the essential building block for cmFANA and the synthesis of the cmFANA oligonucleotide. Attention is now turned to the evolution and application of cmFANA as a sugar-presenting affinity reagent that targets disease-related carbohydrate binding proteins (“CBPs.”). Embodiments of the disclosure below described present an XNA structure that goes beyond established impressions of nucleic acids and displays a high potential ability as a versatile platform technology.
As above discussed, aptamers based on canonical nucleic acids suffer from extreme susceptibility to nuclease-mediated degradation and the lack chemical functionalities that are frequently critical to act as highly specific ligands for protein targets. Embodiments of the disclosure below discussed seek to apply the cmFANA clickable platform technology above disclosed to develop an unnatural nucleic acid affinity reagent through Darwinian evolution systems, as a demonstration of one of the potential applications of cmFANA platform.
The development of an evolvable cmFANA affinity reagent started from the design of an in vitro selection method that circumvent direct reverse-transcription of selected ligand-conjugated cmFANA genetic polymer. Following the DNA-display strategy described above in Example 5, where the carbohydrate-conjugated cmFANA polymer was covalently linked with its single-sequence encoding DNA template, the feasibility of using a DNA template that was encoded with random library sequence is demonstrated. A mock selection was then practiced with successful enrichment of known single-sequence product out from a library pool mixture. The in vitro selection using Concanavalin A (ConA) as CBP target was carried out with three selection cycles and the enriched sequences were analyzed by next-generation high throughput sequencing. Finally, four aptamer candidates were synthesized and functionalized, ready for further binding affinity assays, paving the way for the development of a simple and powerful screening and characterization method for cmFANA-based aptamer.
Embodiments of the disclosure presented in further detail below demonstrate that: First, the genetic information of a majorly structurally modified aptamer was able to be recovered and amplified using the developed in vitro selection cycle. Next, a cmFANA library successfully underwent three rounds of selection and showed obvious enrichment of the target-binding aptamer population. Finally, aptamer candidates from top enriched cluster families were picked and their single-sequenced cmFANA aptamer structures were generated for preliminary binding measurements using flow cytometry.
For the example embodiments disclosed, a bead-based fluorescent assay using a flow cytometer may be performed using the single-stranded single-sequenced cmFANA aptamers below presented to further study the preliminary binding assay and measured the Kd value of the selected aptamers. Furthermore, using the generated cmFANA aptamers, a surface plasmon resonance (SPR) instrument may also be performed for a more comprehensive study of the binding affinity of the aptamer to a protein target.
Additionally, in view of promising binding affinity results presented for an embodiment example using aptamer can-1 shown via a bead-based fluorescent binding assay, more of the 5′-biotinylated aptamer can-1 with and without mannose sugar conjugation, as well as 5′-biotinylated can-1-scrambled single-stranded sequence, which contains a sequence scramble version of can-1 at the N40 cmFANA region, for future Streptavidin-based aptamer immobilizing binding affinity assay using SPR were prepared. All in all, the cmFANA technology demonstrated represents a valuable sequence-defined biopolymer material that possesses the potential to replace the applications of natural genetic polymers, including the ability to undergo laboratory evolution for the generation of a nuclease-resistant, functionality-conjugatable, and target-specific affinity reagent.
In Vitro Selection of Mannose-Conjugated cmFANA Against Con A
In vitro selection of a functionality-conjugated cmFANA library demonstrated successful recovery and enrichment of the genetic information of functionality-conjugated cmFANA sequences that bind to target protein. To begin, the lectin concanavalin A (Con A) was chosen for its commercial availability and easy access to various Con A conjugates. Biotinylated Con A protein was first tested as the target protein for the selection cycle. After generation of the single-stranded DNA template (with N40 random region, the genotype) that would self-hybridized into a hairpin structure, cmFANA was synthesized by Tgo, by extending the hairpin from the 3′-end to obtain a self-hybridized DNA template-cmFANA double-stranded hairpin structure (FIG. 9). Next, since Con A is known to bind to mannose, 2-azidoethyl-α-D-mannose was conjugated to the cmFANA strand as natural mannose sugar mimetic and Con A specific ligand in this experiment. The functionality-conjugated cmFANA strand was then displaced from the DNA genotype by strand displacement process and allowed to fold into corresponding phenotype.
After incubation of the strand-displaced library products with biotinylated Con A, following capture by Streptavidin magnetic particles and ten cycles of buffer wash, the dsDNA genotype was eluted by the Exonuclease-I treatment and was subjected to PCR amplification, strand separation, and hairpin regeneration. Three rounds of selection cycle were performed, which was determined by analyzing the enrichment of recovered post-selection library through qPCR. In a surprising outcome, even functionalized with the Con A-specific ligand, the recovery rate was only around 0.14% for the first round of selection. However, there was then observed a nearly 100-fold enrichment after the second round, and a final 47% recovery rate for the third round (
The starting library as well as post-selection libraries from the first and second round of selection cycle were sent for next-generation sequencing. The FASTQ sequencing data was preprocessed using Python and analyzed using FASTAptameR2.0 to count and rank the unique sequences in each pool. As the selection cycle progressed, the population in unique sequence decreased after each round while total reads increased, which shows consistency with the recovery rate determined by qPCR, indicating the enrichment of the representation of specific post-selection unique sequences and overall lower diversity (
Single-sequenced aptamer candidates were generated by Tgo polymerase conducted primer extension using 5′-biotinylated DNA primer and DNA template with extending region (100 nt template, with 80 nt single-stranded DNA-cmFANA product), as well as FANA nucleotide triphosphates (A, C, and G) and C8-alkyne-FANA UTP. After functionalization with 2-azidoethyl-α-D-mannose, the 5′-biotinylated single-stranded functionality-conjugated cmFANA aptamer product was isolated by immobilization with Streptavidin magnetic bead and then incubation with 250 mM sodium hydroxide to remove DNA template strand (
The single-sequenced functionality-conjugated cmFANA aptamers of four of the selected aptamer sequences from the top four families, which all had >100 fold enrichment compared to the starting library, were synthesized, immobilized on beads, and subject to flow cytometry to measure their fluorescence intensity after incubating with 0 nM to 10 nM Alexa Fluor 647 labeled Con A. Of these four, sequence can-1 (80nt) showed the most fluorescence shift (
All solvents and reagents for chemical synthesis were purchased from commercial sources and used as supplied. All DNA oligonucleotides were purchased from Integrated DNA Technologies. FANA NTPs (FANA ATP, FANA CTP, FANA GTP, FANA UTP) were obtained from Metkinen Chemistry. Next-generation sequencing was performed using the Amplicon-EZ service provided by Genewiz.
DNA-Display Cycle for Carbohydrate-Conjugated cmFANA
The DNA-display cycle was adapted from the similar method described in above Example 5. To begin, 10 μL of 10 μM ssDNA Library 1 (N40), 20 μL of 10×NEBuffer 2, 12 μL of 10 μM Hairpin Regenerating Primer, and 150 μL of water was added to a PCR tube to make a master mix of a total volume of 192 μL. The master mix was separated into two PCR tubes with 96 μL master mix in each PCR tube. The PCR tubes were placed in a thermal cycler and heated with an annealing ramp of 95° C. to 45° C. at a rate of 0.1° C. sec−1 to anneal the ssDNA library template-primer. Then, 2 L of 10 mM DNA dNTPs mix and 2 μL of 5 U/μL DNA polymerase I (Klenow) was added into each PCR tube and the reaction mixtures was incubated for 25 min at 25° C. before directly adding 1.5 μL of 20 U/μL Exo I to each tube and further incubated for 30 minutes at 37° C. and then 20 minutes at 80° C. to denature the enzyme. To perform ethanol precipitation, the reaction mixture of the two PCR tubes were combined in a 1.5 mL Eppendorf tube and added nuclease-free water to a total volume of 300 μL, before added with 40 μL of 3 M sodium acetate pH 5.2 and 1 mL of 100% ethanol, followed by freezing at −80° C. for 30 minutes and centrifugation of the frozen stock 30 min at 21,000×g at 4° C. The supernatant was removed and the pellet of dsDNA regeneration product was resuspended with 95 μL water (reserve 5 μL for PAGE analysis) before combining with 90 μL 2×B & W buffer.
To remove the unwanted biotinylated strand, the dsDNA regeneration product solution (180 μL) was first added to 150 μL of MyOne C1 streptavidin beads (supernatant removed and prewashed three times) and mixed on a rotator for 30 minutes at room temperature. The beads were then captured on a magnet and the supernatant was removed. The beads were washed three times with 150 μL 1×B & W before adding 40 μL of 100 mM NaOH and sit for 4 minutes to elute the non-biotinylated ssDNA. The supernatant containing non-biotinylated ssDNA was then transferred to another tube containing 4 μL of 1 M HCl and 1 μL of 1 M Tris-Cl, pH 8.0 for neutralization. The ssDNA was then quantified by NanoDrop Spectrophotometer and stored at −20° C.
cmFANA synthesis by ssDNA hairpin self-extension reaction was performed in 43 μL reaction volumes containing 35 μL of ssDNA library hairpin previously made, 5 μL of 10×Thermopol buffer, and water to a total volume of 43 μL. The ssDNA was self-annealed to its hairpin formation by heating for 5 minutes at 95° C. and slowly cooled down to 4° C. for 10 minutes. The mixture was then added with 1 μL of 10 mM premixed FANA NTPs (FANA ATP, FANA CTP, and FANA GTP only), 1 μL of 10 mM C8-alkyne-FANA UTP (11), and 5 μL of 8 μM lab expressed Tgo polymerase, followed by incubation for 3 hours at 55° C. to perform cmFANA synthesis. The reaction was then added with water to a total volume of 100 μL and treated with ethanol precipitation, in which 10 μL of 3 M sodium acetate (pH 5.2) and 330 μL of 100% ethanol were added to the solution, followed by freezing at −80° C. for 30 minutes and centrifugation of the frozen stock 30 min at 21,000×g at 4° C. The supernatant was removed and the pellet was resuspended with 22 μL PBS buffer 1×, where 2 μL was reserved for PAGE analysis (10% TBE gel).
Click conjugation of hairpin DNA-cmFANA hybrid with Man-N3 was performed by combining the 20 μL hairpin DNA-cmFANA hybrid solution with 30 μL of 100 mM 2-azidoethyl α-(D)-mannopyranoside in DMSO and 40 μL of 20 mM sodium phosphate buffer (pH 8), followed by addition of 20 μL of a 10 mM, premixed solution of Cu:TBTA=1:1 (prepared with 1 mg CuBr+0.7 mL of 10 mM TBTA in 4:3:1 water:DMSO:tBuOH). For preparing positive control for mock selection experiment, exchange 2-azidoethyl α-(D)-mannopyranoside with Azide-PEG3-biotin. The reaction was incubated for 3 hours at room temperature under nitrogen gas bubbling to displace oxygen in the system. The click reaction mixture was again applied with ethanol precipitation, in which 12 μL of 3 M sodium acetate (pH 5.2) and 400 μL of 100% ethanol were added to the solution, followed by freezing and centrifugation of the frozen stock 30 min at 21,000×g at 4° C. The pellet was resuspended with 22 μL of water and 2 μL was reserved for PAGE analysis (10% TBE gel).
Strand displacement of the self-annealed hairpin was performed by combining 20 μL of the hairpin with 6 μL of 10 μM DNA primer H-FP, 5 μL of 10×Thermopol buffer, and 16 μL of water. After heating for 15 seconds at 65° C. in the thermal cycler, the mixture was quickly added with 1 μL of 10 mM DNA dNTP mix and 2 μL of 8 U/μl Bst2.0 WarmStart DNA Polymerase to a total reaction volume of 50 μL, and the reaction was continued to incubate at 65° C. for another 5 minutes, followed by 60° C. for 5 minutes and then hold at 4° C. Ethanol precipitation (add water to 100 μL, add 10 μL of 3 M sodium acetate pH 5.2 and 330 μL of 100% ethanol, freeze, and centrifuge for 30 min at 21,000×g at 4° C.) was performed after the reaction. The pellet was resuspended with 80 μL of water and the mixture was stored at −80° C. To confirm strand displacement result, 8 μL of the strand displacement solution was added with 1 μL exonuclease I (Exo I) buffer, and 1 μL of 20 U/μl Exo I, and then Incubate for 30 min at 37° C. and reserve on ice for PAGE analysis. Both strand displacement samples before and after exonuclease digestion should be analyzed by PAGE (10% TBE gel) to confirm the success of strand displacement.
PCR amplification of the dsDNA was performed by combining the 1 μL strand displacement product with 2.5 μL of 10 μM H-FP-Bio, 2.5 μL of 10 μM H-RP, 10 μL of 5×Q5 reaction buffer, 1 μL of 10 mM DNA dNTPs mix, 1 μL of 2 U/μL Q5 High-Fidelity DNA Polymerase, and 32 μL of water. PCR conditions were as follow: 98° C. for 30 second; 12 cycles of 98° C. for 10 second, 60° C. for 25 second, 72° C. for 26 second; 72° C. for 2 minutes, and hold at 4° C. To remove excess primers and denature the enzymes, 1 μL of 20 U/μl exonuclease I (Exo I) was directly added to the reactions and incubate in a thermal cycler for 30 minutes at 37° C. and then 20 minutes at 80° C. The four reaction mixtures were combined. Ethanol precipitation (add water to 100 μL, add 10 μL of 3 M sodium acetate pH 5.2 and 330 μL of 100% ethanol, freeze, and centrifuge for 30 min at 21,000×g at 4° C.) was again performed. The pellet of PCR product was resuspended into 180 μL 1×B & W buffer and ready for the next step.
To remove unwanted biotinylated strand, the PCR product solution was first added to 150 μL of MyOne C1 streptavidin beads (supernatant removed and prewashed three times) and mixed on a rotator for 30 minutes at room temperature. The beads were then capture on magnet and the supernatant was removed. The beads were washed three times with 150 μL 1×B & W before adding L of 100 mM NaOH and sit for 4 minutes to elute the non-biotinylated ssDNA. The supernatant containing non-biotinylated ssDNA was then transferred to another tube containing 4 μL of 1 M HCl and 1 μL of 1 M Tris-Cl, pH 8.0 for neutralization. The ssDNA was then quantified by NanoDrop Spectrophotometer and stored at −20° C.
Finally, to regenerate the hairpin sequence to give (N+1)th-generation, the 45 μL of ssDNA product was added with 10 μL of 10×NEBuffer 2, 6 μL of 10 μM Hairpin Regenerating Primer, and 35 μL of water. The ssDNA-primer was annealed in thermal cycler with an annealing ramp of 95° C. to 45° C. at a rate of 0.1° C./sec. Then, 2 μL of 10 mM DNA dNTPs mix and 2 μL of 5 U/μl DNA polymerase I (Klenow) was added and the reaction mixture was incubated for 15 min at 25° C. before directly adding 1.5 μL of 20 U/μl Exo I and further incubated for 30 minutes at 37° C. and then 20 minutes at 80° C. Ethanol precipitation (30 μL of 3 M sodium acetate pH 5.2 and 900 μL of 100% ethanol, freeze, and centrifuge for 30 min at 21,000×g at 4° C.) was again performed. The pellet of dsDNA regeneration product was resuspended into 180 μL 1×B & W buffer. Bead separation was again practiced with exact same procedure described in the previous step to remove unwanted biotinylated strand. The ssDNA hairpin of (N+1)th-generation was quantified by NanoDrop Spectrophotometer and stored at −20° C.
In Vitro Selection with ConA Protein as Target
To begin, 10 μL of 100 μM ssDNA Library 1 (N40), 20 μL of 10×NEBuffer 2, 12 μL of 100 μM Hairpin Regenerating Primer, and 150 μL of water was added to a PCR tube to make a master mix of a total volume of 192 μL. The master mix was separated into two PCR tubes with 96 μL master mix in each PCR tube. The PCR tubes were placed in a thermal cycler and heated with an annealing ramp of 95° C. to 45° C. at a rate of 0.1° C./see to anneal the ssDNA library template-primer. Then, 2 μL of 100 mM DNA dNTPs mix and 2 μL of 5 U/μL DNA polymerase I (Klenow) was added into each PCR tube and the reaction mixtures was incubated for 40 min at 25° C. before directly adding 2 μL of 20 U/μL Exo I to each tube and further incubated for 30 minutes at 37° C. and then 20 minutes at 80° C. to denature the enzyme. To perform ethanol precipitation, the reaction mixture of the two PCR tubes were combined in a 1.5 mL Eppendorf tube and added nuclease-free water to a total volume of 300 μL, before added with 40 μL of 3 M sodium acetate pH 5.2 and 1 mL of 100% ethanol, followed by freezing at −80° C. for 30 minutes and centrifugation of the frozen stock 30 min at 21,000×g at 4° C. The supernatant was removed, and the pellet of dsDNA regeneration product was resuspended with 105 μL water (reserve 5 μL for PAGE analysis) before combining with 100 μL 2×B & W buffer.
To remove unwanted biotinylated strand, the dsDNA regeneration product solution (200 μL) was first added to 200 μL of MyOne C1 streptavidin beads (supernatant removed and prewashed three times with 200 μL 1×B & W buffer) and mixed on a rotator for 30 minutes at room temperature. The beads were then capture on magnet and the supernatant was removed. The beads were washed three times with 200 μL 1×B & W before adding 80 μL of 100 mM NaOH and sit for 4 minutes to elute the non-biotinylated ssDNA. The supernatant containing non-biotinylated ssDNA was then transferred to another tube containing 8 μL of 1 M HCl and 2 μL of 1 M Tris-Cl, pH 8.0 for neutralization. The ssDNA (90 μL total) was then quantified by NanoDrop Spectrophotometer and stored at −20° C.
cmFANA synthesis by ssDNA hairpin self-extension reaction was performed in 50 μL reaction volumes containing 30 μL of ssDNA library hairpin previously made, 5 μL of 10×Thermopol buffer, and water to a total volume of 43 μL. The ssDNA was self-annealed to its hairpin formation by heating for 5 minutes at 95° C. and slowly cooled down to 4° C. for 10 minutes. The mixture was then added with 1 μL of 10 mM premixed FANA NTPs (FANA ATP, FANA CTP, and FANA GTP only), 1 μL of 10 mM C8-alkyne-FANA UTP (compound 11), and 5 μL of 8 μM lab expressed Tgo polymerase, followed by incubation for 3 hours at 55° C. to perform cmFANA synthesis. The reaction was then added with water to a total volume of 100 μL and treated with ethanol precipitation, in which 10 μL of 3 M sodium acetate (pH 5.2) and 330 μL of 100% ethanol were added to the solution, followed by freezing at −80° C. for 30 minutes and centrifugation of the frozen stock 30 min at 21,000×g at 4° C. The supernatant was removed, and the pellet was resuspended with 22 μL PBS buffer 1×, where 2 μL was reserved for PAGE analysis (10% TBE gel).
Click conjugation of hairpin DNA-cmFANA hybrid with Man-N3 was performed by combining the 20 μL hairpin DNA-cmFANA hybrid solution with 30 μL of 100 mM 2-azidoethyl α-(D)-mannopyranoside in DMSO and 40 μL of 20 mM sodium phosphate buffer (pH 8), followed by addition of 20 μL of a 10 mM, premixed solution of Cu:TBTA=1:1 (prepared with 1 mg CuBr+0.7 mL of 10 mM TBTA in 4:3:1 water:DMSO:tBuOH). For preparing positive control for mock selection experiment, exchange 2-azidoethyl α-(D)-mannopyranoside with Azide-PEG3-biotin. The reaction was incubated for 3 hours at room temperature under nitrogen gas bubbling to displace oxygen in the system. The click reaction mixture was again applied with ethanol precipitation, in which 12 μL of 3 M sodium acetate (pH 5.2) and 400 μL of 100% ethanol were added to the solution, followed by freezing and centrifugation of the frozen stock 30 min at 21,000×g at 4° C. The pellet was resuspended with 22 μL of water and 2 μL was reserved for PAGE analysis (10% TBE gel).
Strand displacement of the self-annealed hairpin was performed by combining 20 μL of the functionalized double-stranded hairpin product with 6 μL of 10 μM DNA primer H-FP, 5 μL of 10×Thermopol buffer, and 16 μL of water. After heating for 15 seconds at 65° C. in the thermal cycler, the mixture was quickly added with 1 μL of 10 mM DNA dNTP mix and 2 μL of 8 U/μl Bst2.0 WarmStart DNA Polymerase to a total reaction volume of 50 μL, and the reaction was continued to incubate at 65° C. for another 5 minutes, followed by 60° C. for 5 minutes and then hold at 4° C. Ethanol precipitation (add water to 100 μL, add 10 μL of 3 M sodium acetate pH 5.2 and 330 μL of 100% ethanol, freeze, and centrifuge for 30 min at 21,000×g at 4° C.) was performed after the reaction. The pellet was resuspended with 80 μL of water and the mixture was stored at −80° C. To confirm strand displacement result, 8 μL of the strand displacement solution was added with 1 μL exonuclease I (Exo I) buffer, and 1 μL of 20 U/μl Exo I, and then Incubate for 30 min at 37° C. and reserve on ice for PAGE analysis. Both strand displacement samples before and after exonuclease digestion should be analyzed by PAGE (10% TBE gel) to confirm the success of strand displacement.
The selection process starts by adding 39 μL of the prepared strand displacement product into a clean 1.5 mL Eppendorf tube, then add in 5 μL of 10×PBS, 5 μL of 25 mM MgCl2, 0.5 L of 100 mM CaCl2), 0.5 μL of 100 mM MnCl2, and 0.5 μL of 1% Tween 20 diluted in water. To this solution, 5 μL of 570 mM of biotinylated Con A protein in selection buffer (SB; 1×PBS, 2.5 mM MgCl2, 1 mM CaCl2), 0.1 mM MnCl2, 0.01% Tween 20) was added and allowed to incubate at room temperature with rotation for 1 hr. Then, 2.5 μL of MyOne C1 streptavidin beads (supernatant removed and prewashed three times with SB) was added to the mixture, the solution mixture was further diluted with 150 μL SB and mixed on a rotator for 30 minutes at room temperature to recover aptamer-bind biotinylated Con A. The beads were then capture on magnet and the supernatant was removed. The beads were washed ten times with 200 μL SB before resuspending in 50 μL water. To recover the dsDNA genotype, the following exonuclease digestion was prepared:
PCR amplification of the dsDNA was performed by combining the 0.5 μL recovered post-selection dsDNA library with 2.5 μL of 10 μM H-FP-Bio, 2.5 μL of 10 μM H-RP, 10 μL of 5×Q5 reaction buffer, 1 μL of 10 mM DNA dNTPs mix, 1 μL of 2 U/μL Q5 High-Fidelity DNA Polymerase, and water to a total volume of 50 μL. PCR conditions were as follow: 98° C. for 30 second; 20 cycles of 98° C. for 10 second, 62° C. for 30 second, 72° C. for 26 second; 72° C. for 2 minutes, and hold at 4° C. To remove excess primers and denature the enzymes, 1 μL of 20 U/μl exonuclease I (Exo I) was directly added to the reactions and incubate in a thermal cycler for 30 minutes at 37° C. and then 20 minutes at 80° C. A total of fifty 50 μL PCR mixtures were prepared and combined in a 15 mL conical centrifuge tube. Ethanol precipitation (add 0.25 mL of 3 M sodium acetate pH 5.2 and 7 mL of 100% ethanol, freeze, and centrifuge for 45 min at 5,000×g at 4° C.) was performed. The pellet was dissolved with 300 μL water, followed by purification using MinElute spin columns. The PCR product was eluted with 100 μL water and was added with 100 μL 2×B & W buffer and ready for the next step.
To remove the unwanted biotinylated strand, the PCR product solution was first added to 200 μL of MyOne C1 streptavidin beads (supernatant removed and prewashed three times) and mixed on a rotator for 30 minutes at room temperature. The beads were then capture on magnet and the supernatant was removed. The beads were washed three times with 200 μL 1×B & W before adding L of 100 mM NaOH and sit for 4 minutes to elute the non-biotinylated ssDNA. The supernatant containing non-biotinylated ssDNA was then transferred to another tube containing 4 μL of 1 M HCl and 1 μL of 1 M Tris-Cl, pH 8.0 for neutralization. The ssDNA selection genotype (94nt) was then quantified by NanoDrop Spectrophotometer and stored at −20° C.
Finally, to regenerate the hairpin sequence to give (N+1)th-generation, the 45 μL of ssDNA product was added with 10 μL of 10×NEBuffer 2, 6 μL of 100 μM Hairpin Regenerating Primer, and 35 μL of water. The ssDNA-primer was annealed in thermal cycler with an annealing ramp of 95° C. to 45° C. at a rate of 0.1° C./sec. Then, 2 μL of 50 mM DNA dNTPs mix and 2 μL of 5 U/μl DNA polymerase I (Klenow) was added and the reaction mixture was incubated for 40 min at 25° C. before directly adding 1.5 μL of 20 U/μl Exo I and further incubated for 30 minutes at 37° C. and then 20 minutes at 80° C. Ethanol precipitation (add water to a total volume of 300 μL, then add 30 μL of 3 M sodium acetate pH 5.2 and 900 μL of 100% ethanol, freeze, and centrifuge for 30 min at 21,000×g at 4° C.) was again performed. The pellet of dsDNA regeneration product was resuspended into 180 μL 1×B & W buffer. Bead separation was again practiced with exact same procedure described in the previous step to remove unwanted biotinylated strand. The ssDNA hairpin of (N+1)th-generation was quantified by NanoDrop Spectrophotometer and stored at −20° C.
A second-generation ssDNA hairpin self-extension reaction was performed as described above and an aliquot of the extended hairpin DNA-cmFANA hybrid product was analyzed by PAGE (10% TBE gel) to confirm the success of the DNA-display cycle.
A second-generation ssDNA hairpin self-extension reaction was performed as described above and an aliquot of the extended hairpin DNA-cmFANA hybrid product was analyzed by PAGE (10% TBE gel) to confirm the success of the DNA-display cycle.
The next-generation sequencing of the library was performed according to the Amplicon-EZ workflow provided by Genewiz. Briefly, the ssDNA selection genotype (94nt) of after the first and second rounds of selection were further amplified by PCR with extended forward (Amplicon_FP_XL) and reverse primers (Amplicon_RP_XL) to add extra 56 bp to the dsDNA amplicon sample, making the length of the amplicon sequence 150 bp without the adapters, as well as to install the adapter and a spacer such that the length of the PCR product is 215 bp (dsDNA amplicon sample 2) required for Genewiz Amplicon-EZ sequencing. The sequencing results and FASTQ files were analyzed by a python code that provided the total number of reads, read length, number of unique sequences, and visualization (
Single-stranded cmFANA were generated by first preparing a master mix solution in a 1.5 mL Eppendorf tube containing 20 μL of 100 μM BindConA_primer_Bio, 20 μL of 100 μM (T_BindConA_can-1, or T_BindConA_can-2, or T_BindConA_can-3, or T_BindConA_can-4), 200 μL of 10×ThermoPol buffer, and 560 μL ultra-pure water (800 μL total). The 1.5 mL Eppendorf tube containing the mast mix was placed on a hot plate with temperature set at 90° C. for 10 minutes and cooling on ice for 15 minutes. The master mix was transfer to a larger tube before adding 200 μL of 1 mM of FANA NTPs (A, C, and G), 200 μL of 1 mM C8-alkyne-FANA UTP (compound 11, above described), 400 μL of lab expressed Tgo polymerase (a final concentration of 1 μM), and water to a total volume of 2 mL. The mixture was separated into four 1.5 mL Eppendorf tubes (500 μL each) and placed on hotplate with temperature set at 55° C. Primer-extension reactions were performed for 3 hours at 55° C. before placing on ice.
The primer extension reaction mixtures were collected in a 15 mL centrifuge tube and added with 0.25 mL of 3 M sodium acetate (pH 5.2) solution and 6.875 mL of 100% ethanol, followed by freezing at −80° C. for 30 minutes or liquid nitrogen for 5 minutes. The frozen stock was then centrifuged for 30 min at 4000×g at 4° C. to precipitate the DNA-cmFANA hybrid. The pellet was dissolved with 300 μL water, followed by purification using MinElute spin columns where the primer extension product was eluted with 90 μL of TE buffer (10 mM Tris-Cl, 1 mM EDTA, pH 8). The collected primer extension product was again applied with ethanol precipitation, in which 20 μL of 3 M sodium acetate (pH 5.2) and 600 μL of 100% ethanol were added to the solution, followed by freezing and centrifugation of the frozen stock 30 min at 21,000×g at 4° C. The precipitated DNA-cmFANA hybrid was resuspended in 20 μL 1×PBS buffer.
To perform click conjugation, the 20 μL DNA-cmFANA hybrid solution was added with 20 μL of 100 mM 2-azidoethyl-α-(D)-mannopyranoside in DMSO and 40 μL 20 mM sodium phosphate buffer (pH 8) in a 1.5 mL Eppendorf tube. The cap of the tube was cut off and sealed with a rubber septum, and the system was purged with nitrogen gas for 15 minutes. The click reaction was initiated by adding 20 μL of a 10 mM, premixed solution of Cu:TBTA=1:1 (prepared with 1 mg CuBr+0.7 mL of 10 mM TBTA in 4:3:1 water:DMSO:tBuOH), followed by nitrogen gas purge for 5 minutes and incubated for 4 hours. The click reaction mixture was then applied with ethanol precipitation, in which 10 μL of 3 M sodium acetate (pH 5.2) and 330 μL of 100% ethanol were added to the solution, followed by freezing and centrifugation of the frozen stock 30 min at 21,000×g at 4° C. The sugar-conjugated DNA-cmFANA hybrid pellet was resuspended with 200 μL 1×B & W buffer (5 mM Tris-HCl pH 7.5, 0.5 mM EDTA, 1 M NaCl) and ready for the next step.
To perform bead separation, 200 μL MyOne C1 streptavidin beads was added to a new 1.5 mL Eppendorf tube. The beads were captured on the side of the tube with a magnet and the supernatant was removed. The beads were washed three times with 200 μL 1×B & W. The click product solution was added to the beads and mixed on a rotator for 30 minutes at room temperature. Then, the beads were captured, and the supernatant was removed. The beads with sugar-conjugated DNA-cmFANA hybrid were washed three times with 200 μL 1×B & W, and then treated twice with 50 μL of freshly-prepared 0.25 M NaOH solution to remove non-biotinylated strand and generate single-stranded sugar-cmFANA on the beads. The beads were again captured, and the supernatant was removed. Finally, the beads with cmFANA aptamer were washed again with 1 mL 1×PBS buffer before resuspending in 100 μL 1×PBS buffer.
Around 7-10×106 bead particles (biotinylated functionalized aptamer immobilized on Dynabeads MyOne Streptavidin C1) were incubated with varying concentrations of fluorescently labeled Con A (0-10 nM) in SB for 1 hour on a rotator at room temperature. The beads were washed three times with 200 μL 1×BD Sheath Solution after incubation and resuspended in 10 μL 1×BD Sheath Solution. From this, 3 μL of the beads were transferred to 300 μL 1×BD Sheath Solution for flow cytometry. The beads were analyzed using the BD Accuri C6 flow cytometer, and the mean fluorescence and/or percentage of bound particles were measured in the relevant fluorescence channel(s).
U*CG U*CA AAG AU*G CCU* AAG AU*C
U*CG A C*U* GU*G CAA ACG GC
U*AU*A GG-3′
U*AU* GGU* U*U*C AAC ACG U*GC
U*A U* U*GA CAC U GA GGC AAA U*A
U* AAG G CU*U* GU*G CAA ACG GC
U*AU*A GG-3′
It is understood that, while particular embodiments have been illustrated and described, various modifications may be made thereto and are contemplated herein. It is also understood that the disclosure is not limited by the specific examples provided herein. The description and illustration of embodiments and examples of the disclosure herein are not intended to be construed in a limiting sense. It is further understood that all aspects of the disclosure are not limited to the specific depictions, configurations or relative proportions set forth herein, which may depend upon a variety of conditions and variables. Various modifications and variations in form and detail of the embodiments and examples of the disclosure will be apparent to a person skilled in the art. It is therefore contemplated that the disclosure also covers any and all such modifications, variations and equivalents.
This application claims the benefit under 35 U.S.C. 119(e) of previously filed U.S. Provisional Patent Application Ser. No. 63/499,476, titled: Click Handle-Modified Deoxy-Fluoroarabino Nucleic Acid as a Synthetic Genetic Polymer Capable of Post-Polymerization Functionalization, filed: 1 May 2023, which is incorporated herein by reference in its entirety for all purposes.
This invention was made with government support under grant DP2-HG011027 awarded by the National Institutes of Health. The government has certain rights in the invention.
| Number | Date | Country | |
|---|---|---|---|
| 63499476 | May 2023 | US |
