Not applicable.
The emerging field of synthetic genetics provides an exciting opportunity to explore the structural and functional properties of synthetic genetic polymers by in vitro selection. However, achieving the goal of artificial genetics requires the ability to synthesize unnatural nucleic acid substrates (“XNA”s), such as threose-nucleic acids (“TNAs”), that are not otherwise available. Limiting this process is the availability of enzymes and conditions that allow for the storage and propagation of genetic information present in unnatural nucleic acid polymers such as TNAs.
Described herein are methods, compositions, and systems for replicating and evolving threose nucleic acids.
Accordingly, in a first aspect disclosed herein is a method for synthesizing a threose nucleic acid, comprising: contacting a single stranded DNA template hybridized to a primer with a DNA polymerase comprising an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO:1 in the presence of tTTP, tGTP, tATP; and (i) dCTP; or (ii) a combination of tCTP and dCTP; and incubating at a temperature suitable for polymerization by the DNA polymerase to obtain a threose nucleic acid.
In some embodiments of the first aspect, the DNA polymerase is in the presence of tTTP, tGTP, tATP, and dCTP. In some embodiments, where the DNA polymerase is in the presence of tTTP, tGTP, tATP, and dCTP, the contacting step is done in the substantial absence of tCTP. In some embodiments, a threose nucleic acid is provided that is generated according to the just-mentioned method, where the synthesis reaction includes dCTP and is substantially free of tCTP.
In some embodiments of the first aspect, the DNA polymerase is in the presence of tATP, tTTP, tGTP, and a combination of tCTP and dCTP.
In some embodiments of the first aspect, the DNA polymerase comprises the amino acid sequence of SEQ ID NO:1.
In some embodiments of the first aspect, the single stranded DNA template sequence is restricted to the nucleotides dA, dC, and dT.
In other embodiments of the first aspect, the single stranded DNA template sequence comprises 7-deaza-dGTP instead of dGTP.
In a second aspect disclosed herein is a method for reverse transcribing a threose nucleic acid, comprising contacting a threose nucleic acid template with a SuperScript II reverse transcriptase in the presence of a primer and dNTPs, dNTP analogs, or a combination thereof to obtain a threose nucleic acid reverse-transcription mix, and incubating the mix at a temperature suitable for SuperScript II reverse transcriptase activity to obtain a cDNA copy of the threose nucleic acid template, where the threose nucleic acid template comprises deoxycytidine.
In a third aspect disclosed herein is a for molecular evolution of threose nucleic acids, where the method includes the steps of: (i) providing a DNA template library comprising diverse DNA template sequences; (ii) hybridizing the template library with one or more complementary primer sequences; (iii) incubating the hybridized template library with a DNA polymerase comprising an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO:1 in the presence of tTTP, tGTP, tATP; and (i) dCTP; or (ii) a combination of tCTP and dCTP; and incubating at temperature suitable for polymerization by the DNA polymerase to obtain a cTNA library; (iv) subjecting the cTNA library to a selection assay to obtain at least one or more selected cTNAs; and (v) incubating the one or more selected cTNAs with a primer, a SuperScript II reverse transcriptase, and dNTPs at a temperature suitable for SuperScript II reverse transcriptase activity to obtain to obtain a selected DNA template library.
In some embodiments of the third aspect, the diverse DNA template sequences are restricted to the nucleotides dA, dC, and dT.
In some embodiments of the third aspect, the selection assay in step (iv) comprises selection of one or more cTNAs based on affinity for a ligand. In some embodiments the selection assay in step (iv) comprises selection of one or more cTNAs based on a catalytic activity. In other embodiments the selection assay in step (iv) comprises selection of one or more cTNAs based on fluorescence emission.
In some embodiments of the third aspect, step (iii) is done in the substantial absence of tCTP.
In some embodiments of the third aspect, the DNA template library comprises DNA templates comprising 7-deaza-dGTP instead of dGTP.
In a fourth aspect disclosed herein is a TNA transcription system comprising a single stranded DNA template, a DNA polymerase comprising an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO:1, tTTP, tGTP, tATP; and (i) dCTP; or (ii) a combination of tCTP and dCTP.
In some embodiments of the fourth aspect the TNA transcription system comprises dCTP, but is substantially free of tCTP.
In some embodiments of the fourth aspect, the single stranded DNA template comprises 7-deaza-dGTP instead of dGTP.
In a fifth aspect disclosed herein is a TNA reverse transcription system comprising a TNA template, a SuperScript II reverse transcriptase, and dNTPs; wherein the TNA template comprises dC.
The present invention will be better understood and features, aspects and advantages other than those set forth above will become apparent when consideration is given to the following detailed description thereof. Such detailed description makes reference to the following drawings, wherein:
Disclosed herein are methods, compositions and systems for replication and in vitro evolution of TNAs based on the unexpected finding that certain TNA synthesis conditions, as described herein, permit the efficient and faithful synthesis of XNAs from DNA templates and their reverse transcription into cDNAs using known polymerases.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although any methods and materials similar to or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described herein.
In describing the embodiments and claiming the invention, the following terminology will be used in accordance with the definitions set out below.
As used herein, “about” means within 5% of a stated range within the relevant parameter.
As used herein, “TNA” or “TNAs” refer to nucleic acids having a backbone composed primarily of α-
As used herein, “tNTPs” refer to threose nucleotide triphosphates.
As used herein, “tNTP analog” refers to a threose nucleotide triphosphate having a modified base moiety.
With respect to the amino acid sequence homology of polypeptides described herein, one of ordinary skill in the art will appreciate that structural and functional homology of two or polypeptides generally includes determining the percent identity of their amino acid sequences to each other. Sequence identity between two or more amino acid sequences is determined by conventional methods. See, for example, Altschul et al., (1997), Nucleic Acids Research, 25(17):3389-3402; and Henikoff and Henikoff (1982), Proc. Natl. Acad. Sci. USA, 89:10915 (1992). Briefly, two amino acid sequences are aligned to optimize the alignment scores using a gap opening penalty of 10, a gap extension penalty of 1, and the “BLOSUM62” scoring matrix of Henikoff and Henikoff (ibid.). The percent identity is then calculated as: ([Total number of identical matches]/[length of the longer sequence plus the number of gaps introduced into the longer sequence in order to align the two sequences])(100).
Described herein are methods for efficient synthesis of a TNA from a DNA template. In various embodiment the methods include the steps of contacting a single stranded DNA template hybridized to a primer with a DNA polymerase comprising an amino acid sequence at least 95% (e.g., 97%, 98%, 99%, or 100%) identical to the amino acid sequence of Therminator™ DNA polymerase known under the tradename Therminator™ polymerase (New England Biolabs, MA) in the presence of tTTP, tGTP, tATP; and (i) dCTP; or (ii) a combination of tCTP and dCTP. The amino acid sequence of Therminator™ DNA polymerase is shown below as SEQ ID NO:1.
In some embodiments, the DNA polymerase comprises an A485L point mutation relative to the amino acid sequence of the 9° N DNA polymerase and is greater than about 95% identical to the amino acid sequence of Therminator™ DNA polymerase (Therminator™ DNA polymerase), e.g., about 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of Therminator™ DNA polymerase. In one embodiment, the DNA polymerase to be used comprises the amino acid sequence of SEQ ID NO:1. Typically, TNA synthesis using the Therminator™ polymerase is carried out at about 50° C. to about 60° C. In some embodiments, the TNA synthesis reaction is carried out at about 55° C. Suitable concentrations of tNTPs range from about 20 μM to about 100 μM, e.g., about 25, 30, 35, 40, 50, 60, 70, 80, or another concentration of tNTPs from about 20 μM to about 100 μM.
In some embodiments, the single stranded DNA template to be used in the method comprises a sequence that is restricted to the nucleotides dA, dC, and dT. While not wishing to be bound by theory, it is believed that by limiting single stranded templates to sequences containing these three nucleotides, the fidelity of the sequence transcribed into TNAs is significantly increased as described herein. In other embodiments, the single stranded DNA template to be used comprises 7-deaza-dGTP instead of dGTP to reduce or eliminate dG-tG mispairing, and thereby increase replication fidelity. Also encompassed herein are heteropolymeric TNAs generated by the above-described method, which include tA, tT, tG, and dC.
Also described herein is method for reverse transcribing a TNA. In various embodiments, a TNA is reverse transcribed by a method that includes: contacting a TNA template that contains dCTP with a SuperScript II reverse transcriptase in the presence of a primer and dNTPs, and incubating the resulting mix, at a temperature suitable for SuperScript II reverse transcriptase activity, to obtain a cDNA copy of the TNA template.
Typically the reverse transcription reaction using the SuperScript II reverse transcriptase is carried out at a temperature of about 37° C. to about 45° C. In some embodiments, the TNA reverse transcription reaction is carried out at 42° C.
Also disclosed herein is a method for molecular evolution of threose nucleic acids, which includes the steps of: (i) providing a DNA template library containing diverse DNA template sequences; (ii) hybridizing the template library with one or more complementary primer sequences; (iii) incubating the hybridized template library with a DNA polymerase comprising an amino acid sequence at least 95% (e.g., 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:1 in the presence of tTTP, tGTP, tATP, and dCTP, and incubating at a temperature suitable for polymerization by the DNA polymerase to obtain a cTNA library; (iv) subjecting the cTNA library to a selection assay to obtain at least one or more selected cTNAs; and (v) incubating the one or more selected cTNAs with a primer, a SuperScript II reverse transcriptase, and dNTPs at a temperature suitable for SuperScript II reverse transcriptase activity to obtain a selected DNA template library.
In some embodiments, the diverse DNA template sequences are restricted to dA, dC, and dT. In some embodiments, the DNA template sequences contain 7-deaza-dGTP instead of dGTP.
TNAs can be selected from a cTNA library in step (iv) based on a number of different criteria and assays depending on a desired functionality or endpoint for the TNAs being generated. Accordingly, in some embodiments the selection assay in sep (iv) includes selection of one or more cTNAs from the cTNA library based on affinity for a ligand. Examples of suitable affinity assays known in the art include, but are not limited to, aptamer affinity chromatography, systematic evolution of ligands by exponential enrichment (SELEX), and kinetic capillary electrophoresis. In other embodiments, selection of one or more cTNAs from the cTNA library is based on a catalytic activity. Methods for assaying and selecting catalytic activities, e.g., ribozyme activities, are known in the art as described in, e.g., Link et al. (2007), Biol Chem 388(8):779-786. In some embodiments, one or more cTNAs are selected based on a desired fluorescence emission. See, e.g., Paige et al (2011), Science, 333(6042):642-646.
In the various methods described herein, hybridization between a primer and its target sequence is generally carried out under high stringency conditions under which the primer is annealed with its complementary template sequence at a temperature approximately 5° C. below the primer's melting temperature Tm.
Also described herein are TNA transcription systems. In various embodiments a TNA transcription system includes the following components: a single stranded DNA template, a DNA polymerase comprising an amino acid sequence at least 95% identical to the amino acid sequence of Therminator™ DNA polymerase, tTTP, tGTP, tATP; and (i) dCTP; or (ii) a combination of tCTP and dCTP.
Also disclosed herein are TNA reverse transcription systems. Generally a TNA reverse transcription system, as described herein, includes: a TNA template comprising dC, a SuperScript II reverse transcriptase, and dNTPs.
The DNA primer P1 was 5′-end labeled by incubation in the presence of [γ-32P] ATP with T4 polynucleotide kinase for 1 h at 37° C. The 32P labeled primer was annealed to the DNA template (Table 1) in 1× ThermoPol buffer [20 mM Tris-HCl, 10 mM (NH4)2SO4, 10 mM KCl, 2 mM MgSO4, 0.1% Triton X-100, pH 8.8 at 25° C.] by heating at 95° C. for 5 min and cooling on ice. Primer extension reactions were performed in 10 μl volumes containing 100 μM tNTPs (or a combination of defined tNTP and dNTP mixtures), 500 nM primer-template complex, 1 mM DTT, 100 μg/ml BSA, 1.25 mM MnCl2 and 0.1 U/μl Therminator DNA polymerase. Reactions were initiated by adding the tNTP substrates to a solution containing all other reagents and heating the mixture for 1 h at 55° C. Primer extension products were analyzed by 20% denaturing polyacrylamide gel electrophoresis, imaged with a phosphorimager, and quantified using ImageQuant software (GE Healthcare Biosciences, Pittsburgh, Pa.).
We began by chemically synthesizing each of the α-
To address these concerns, we examined the efficiency of tATP as a substrate for Therminator™ DNA polymerase. As illustrated in
The 32P-labelled DNA primer P3 was annealed to a TNA template in 1× First Strand buffer [50 mM Tris-HCl, 75 mM KCl, 3 mM MgCl2 (pH 8.3 at 25° C.)] by heating at 95° C. for 3 min and cooling on ice. Primer extension reactions contained 500 μM dNTPs, 100 nM primer-template complex, 10 mM DTT, 3 mM MgCl2, 1.5 mM MnCl2 and 10 U/μl SuperScript II reverse transcriptase. Reactions were initiated by adding the enzyme to a solution containing all other reagents, and heating the reaction mixture for 1 h at 42° C. Primer extension products were analyzed by 20% denaturing polyacrylamide gel electrophoresis, imaged with a phosphorimager, and quantified using ImageQuant software (GE Healthcare Biosciences, Pittsburgh, Pa.).
In order to generate a sufficient amount of TNA template to be used in a reverse transcription reaction, TNA synthesis reactions were performed as described above in Example 1 using unlabeled DNA primer P2 in a 400 μl reaction. After incubation for 1 hour at 55° C., the TNA product was separated from the DNA template by 10% denaturing polyacrylamide gel electrophoresis. The band corresponding to the TNA product was excised and the gel slices were electroeluted for 2 hours at 200V. The final solution was ethanol precipitated and quantified by UV absorbance.
32P-labelled DNA primer P3 was annealed to the TNA template in 1× First Strand buffer [50 mM Tris-HCl, 75 mM KCl, 3 mM MgCl2 (pH 8.3 at 25° C.)] by heating at 85° C. for 3 min and cooling on ice. Primer extension reactions contained 500 μM dNTPs, 100 nM primer-template complex, 10 mM DTT, 3 mM MgCl2, 1.5 mM MnCl2 and 10 U/μ1 SuperScript II™ reverse transcriptase. Reactions were initiated by adding the enzyme to a solution containing all other reagents, and heating the reaction mixture for 1 h at 42° C. Primer extension products were analyzed by 20% denaturing polyacrylamide gel electrophoresis, imaged with a phosphorimager, and quantified using ImageQuant software (GE Healthcare Biosciences, Pittsburgh, Pa.). As shown in
The in vitro selection of XNA molecules in the laboratory requires enzymes that can transcribe and reverse transcribe XNA polymers with high efficiency and fidelity. In a recent new advance, Pinheiro et al. used a compartmentalized self-tagging strategy to evolve several polymerases with XNA activity. One of these enzymes, RT521, was created from TgoT, a variant of the replicative polymerase from Thermococcus gorgonarius, for the ability to reverse transcribe HNA back into DNA. In addition to HNA reverse transcriptase activity, RT521 was also found to reverse transcribe other XNA polymers with varying degrees of efficiency. This included arabinonucleic acids, 2′-fluoro-arabinonucleic acids and TNA35. The observation that RT521 could reverse transcribe portions of a TNA template into DNA led us to consider this enzyme as a possible polymerase for the replication TNA polymers in vitro.
To examine the activity of RT521 as a TNA-dependent DNA polymerase, we performed a polymerase activity assay to access the ability for RT521 to reverse transcribe long TNA templates into DNA. Because it is not possible to generate long TNA polymers by solid-phase synthesis, we transcribed a DNA template into TNA using Therminator™ DNA polymerase (
In an attempt to improve the efficiency of TNA-dependent DNA polymerization by RT521, we explored a variety of conditions that have proven helpful in the past. To our surprise, varying the reaction time, salt conditions, and enzyme concentration all proved ineffective. Even the addition of manganese ions, which is known to relax the specificity of many DNA polymerases, inhibited the reaction. The presence of diaminopurine residues in the TNA template also failed to improve the yield of full-length product. The limited TNA synthesis observed in these reactions may reflect an unknown sequence specificity of the enzyme. Alternatively, it is also possible that the sample of RT521 used in our study was less active than the sample used in the original study by Pinheiro et al.
However, close examination of the previous reverse transcription reaction revealed a substantial amount of truncated product, suggesting that RT521 may require further optimization before it can function as an efficient TNA-dependent DNA polymerase.
Recognizing the limitations of RT521, we pursued other enzymes as possible candidates for a TNA reverse transcriptase. In this regard, we have previously screened a wide range of natural and mutant DNA and RNA polymerases for the ability to copy a short chimeric DNA-TNA template containing nine TNA residues in the template region. This study identified the reverse transcriptases MMLV and SuperScript II (SSII) as efficient TNA-dependent DNA polymerases that could copy a short TNA template into DNA with ˜30% full-length product conversion observed after an incubation of 1 hour at 42° C. To determine whether these enzymes could be made to function on longer TNA templates, we explored a range of conditions that would allow the enzymes to copy a 90-nt TNA template back into DNA. Since it was possible that diaminopurine would enhance the efficiency of reverse transcription, we performed the polymerase activity assay on in vitro transcribed TNA containing either adenine or diaminopurine nucleotides in the template strand. Preliminary studies indicated that SSII functioned with greater efficiency and reproducibility than MMLV. Subsequent optimization of this reaction led us to discover conditions that enabled SSII to reverse transcribe the entire TNA template into DNA (
To assess the efficiency of SSII-mediated reverse transcription, we performed a time course analysis to compare the rate of product formation as a function of template composition. Analysis of product formation over time revealed that reverse transcription of the adenine-containing template is complete in 1 hour, while the diaminopurine-containing template required nearly 2 hours to copy the TNA template into DNA (
DNA sequencing was used to measure the fidelity for the overall process of TNA replication and cloning. DNA templates of a defined sequence were transcribed into TNA as described above using primer P2. Primer P2 has an internal reference nucleotide that is designed to unambiguously distinguish cDNA obtained from TNA replication from the starting DNA template. The DNA-TNA heteropolymer was purified by denaturing polyacrylamide gel electrophoresis, and reverse transcribed back into DNA. The resulting cDNA strand was amplified by PCR using primers that matched the outside region of P2 (i.e. P3 and P4). AccuPrime Taq High Fidelity DNA Polymerase was used to minimize possible mutations caused by PCR. Additionally, separate PCR reactions were performed on purified TNA templates to confirm that the PCR product was amplified from cDNA generated in TNA reverse transcription. PCR products were cloned into pJET1.2 vector, transformed into E. coli XL1-Blue competent cells, grown to log phase, the vector was isolated using PureYield™ Plasmid Miniprep System (Promega, Madison, Wis.). Isolated vectors were sequenced at the ASU DNA Sequencing Facility.
We measured the fidelity of TNA replication by sequencing the cDNA product of the reverse transcription reaction after amplification by PCR. This fidelity assay measures the aggregate fidelity of a complete replication cycle (DNA→TNA→DNA), which is operationally different than the more restricted view of fidelity as the accuracy of a single-nucleotide incorporation event. The fidelity determined by this assay is the actual accuracy with which full-length TNA is synthesized and reverse transcribed, and therefore reflects the combined effects of nucleotide misincorporation, insertions and deletions (indel), and any mutations that occur during PCR amplification and cloning.
Several controls were implemented to ensure that the sequencing results represented the true fidelity of TNA replication (
We began by measuring the fidelity of TNA replication for the adenine-containing template used in the reverse transcription assay with SSII. This template, referred to as 4NT.3G, derives from a single sequence that was present in the L3 library30. The L3 library was designed to overcome the problem of polymerase stalling at G-repeats by reducing the occurrence of G residues in the template to 50% the occurrence of A, C, and T. Our earlier work on TNA transcription established the L3 library as an efficient design strategy for generating pools of full-length TNA molecules. While TNA replication on 4NT.3G resulted in an overall fidelity that was comparable with other XNA replication systems (96.4%), detailed analysis of the mutation profile indicated that G→C transversions account for 90% of the genetic changes (
While the precise molecular details of the G→C transversion remain unknown, our results suggest that base stacking plays an important role in the misincorporation of tGTP opposite deoxyG in the template. This prediction is supported by the fact that the frequency of dG:tG mispairing increases 10-fold when G-nucleotides in the template are preceded by pyrimidine residues, indicating that purine residues (A or G) on the growing TNA strand stabilize the incoming tGTP substrate via base stacking interactions. To better understand the problem of dG:tG mispairing, we measured the fidelity of TNA replication using different combinations of template and substrate (
Substituting tGTP for dGTP and assaying a template devoid of C residues produced similar results with 97.5% and 98.2% fidelity, respectively. The mutational profiles obtained under these conditions provide evidence that dG:tG mispairing can be overcome by engineering DNA templates to avoid the problem of nucleotide misincorporation.
In an effort to further improve the fidelity of TNA replication, we examined the mutational profile of two different types of DNA templates that were designed for high fidelity replication. The first template, 3NT.ATC, contained a central region of 50-nts that was composed of a random distribution of A, T, and C residues that were flanked by two 20-nt fixed-sequence primer-binding sites. This sequence derived from library L2, which we used previously to evolve a TNA aptamer to human thrombin. We found that the L2 library transcribes and reverse transcribes with very high efficiency as judged by the amount of starting primer that is extended to full-length TNA product and the absence of any significant truncated products (
It is hypothesized that dG:tG mispairing occurs through a Hoogsteen base pairing mode rather than the traditional Watson-Crick mode. Nitrogen-7 is critically important for the formation of the Hoogsteen base pair and removal of nitrogen-7 from guanosine in either the templating G residues or substrate tGTPs would eliminate that base pairing mode and prevent dG:tG mispairing. The 4NT.9G template and an unbiased four nucleotide library containing equal amounts of dC, dG, dA, and dT containing 7-deaza-dG in place of dG were generated by asymmetric PCR. The 7-deaza-dG asymmetric PCR reaction is identical to normal asymmetric PCR reactions aside from dGTP being replaced by 7-deaza-dGTP in an equal concentration. PCR reaction products were purified by polyacrylamide gel electrophoresis and used in TNA extension assays and fidelity measurements identical to above. We found that the replication fidelity of four nucleotide templates improved from 96.4% to 99.6%. The fidelity was on par with templates containing only dA, dT, and dC. Additionally, TNA transcription of the four nucleotide library measured by primer extension and gel electrophoresis showed equivalent full length product to three nucleotide libraries.
DNA, RNA, and TNA oligonucleotide substrates (1 nmol) were incubated for up to 72 hours at 37° C. in presence of RQ1 DNase or RNase A using the manufacture's recommended conditions. The DNase reaction contained 1× RQ1 DNase reaction buffer [40 mM Tris-HCl, 10 mM MgSO4, 1 mM CaCl2, pH 8.0] and 0.2 U/μ1 of RQ1 RNase-free DNase in reaction volume of 10 μl. The RNase reaction contained 50 mM NaOAc (pH 5.0) and 0.24 μg/μl RNase A in a reaction volume of 10 μl. Time course reactions were performed by initiating multiple reactions in parallel, removing individual tubes at defined time points, quenching the reaction by the addition of 7 M urea and 20 mM EDTA, storing the quenched reactions at −20° C. until the time course was complete. Time-dependent oligonucleotide stability against DNase or RNase was analyzed by 20% denaturing polyacrylamide gel electrophoresis, and visualized by UV shadowing.
RNA template T1 was synthesized by in vitro transcription using T7 RNA polymerase. After purification by denaturing PAGE, the RNA transcript was dephosphorylated using calf intestinal alkaline phosphatase, and then 5′-end labeled by incubation in the presence of [γ-32P] ATP with T4 polynucleotide kinase. 32P-labeled RNA template T1 (25 pmol) was incubated with a complementary DNA oligonucleotide probe S2 or TNA oligonucleotide probe S3 (50 pmol) for 15 min at 37° C. Each reaction contained 44 μl of reaction buffer [10 mM Tris-HCl, 25 mM KCl, 1 mM NaCl, and 0.5 mM MgCl2, pH 7.5] and 6 μl RNase H (5 U/μl). Control tubes received buffer in place of enzyme. Aliquots were removed at the indicated time points, quenched by the addition of 7 M urea and 20 mM EDTA, and analyzed by 20% denaturing polyacrylamide gelelectrophoresis.
A major goal of synthetic genetics is to create nuclease resistant aptamers and enzymes that function in complex biological environments. To evaluate the nuclease stability of TNA, we synthesized a synthetic TNA 16-mer having the sequence 3′-AAAATTTATTTATTAA-2′ (SEQ ID NO:14) by solid phase phosphoramidite chemistry. The TNA oligonucleotide was tested for nuclease stability against the enzymes RQ1 DNase and RNase A, which degrade DNA and RNA, respectively. In both cases, 1 nmol of the TNA sample was incubated at 37° C. in a reaction buffer of 40 mM Tris-HCl, 10 mM MgSO4, 1 mM CaCl2 (pH 8.0) for the DNase digestion and a reaction buffer of 50 mM NaOAc (pH 5.0) for the RNase digestion. The samples were removed at specified time points, quenched with urea, and analyzed by denaturing polyacrylamide gel electrophoresis. As a control, synthetic DNA and RNA strands with the same sequence were incubated with their respective nuclease and analyzed under time frames that coincided with their degradation. As expected, the DNA sample is rapidly degraded in the presence of RQ1 DNase and exhibited a half-life of ˜30 minutes (
The invention has been described in connection with what are presently considered to be the most practical and preferred embodiments. However, the present invention has been presented by way of illustration and is not intended to be limited to the disclosed embodiments. Accordingly, those skilled in the art will realize that the invention is intended to encompass all modifications and alternative arrangements within the spirit and scope of the invention as set forth in the appended claims.
This application claims priority to U.S. provisional patent application 61/748,834 filed on Jan. 4, 2013, which incorporated by reference herein in its entirety.
Filing Document | Filing Date | Country | Kind |
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PCT/US2014/010062 | 1/2/2014 | WO | 00 |
Number | Date | Country | |
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61748834 | Jan 2013 | US |