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US-12618795-B2 - Methods for asymmetric semi-nested isothermal nucleotide amplification

US12618795B2US 12618795 B2US12618795 B2US 12618795B2US-12618795-B2

Abstract

Aspects of the present disclosure relate to methods, compositions, and systems for asymmetric semi-nested isothermal nucleotide amplification (ANINA) for the amplification of single-stranded oligonucleotides. In some aspects, the methods, compositions, and systems herein do not require thermal melting and may be used in a point-of-need setting. In some aspects, the methods feature amplification of a target region and production of single stranded amplicons including the target region.

Inventors

  • Anindita ROY

Assignees

  • SEEK LABS, INC.

Dates

Publication Date
20260505
Application Date
20220407

Claims (20)

  1. 1 . A method of asymmetric semi-nested isothermal nucleotide amplification for producing single-stranded oligonucleotide amplicons comprising a target region of a template nucleic acid molecule, said method comprising: a) introducing to a sample comprising the template nucleic acid molecule: i) a set of primers comprising: A) a first primer (P1) comprising a first template nucleotide sequence, wherein the entire first template nucleotide sequence is complementary to a first complementary binding region (CSBR1), wherein said P1 is 5′ to the target region, and wherein said CSBR1 is on a strand opposite to the target region; B) a second primer (P2) comprising a second template nucleotide sequence, wherein the entire second template nucleotide sequence is complementary to a second complementary binding region (CSBR2), wherein P2 is (i) 5′ to the target region or (ii) 5′ to the target region and comprises a portion of the target region, wherein said CSBR2 is on a strand opposite to the target region, and wherein P1 is at least partially 5′ to P2; and C) a third primer (P3) comprising a third template nucleotide sequence, wherein the entire third template nucleotide sequence is complementary to a target strand binding region (TSBR) of the target strand, wherein the TSBR comprises (i) at least a portion of the target region, (ii) a portion 3′ to the target region, or (iii) any combination of (i) and (ii), wherein the ratio of P1:P2:P3 in the set of primers is (1-10):(10-200):(1-20); and ii) a solution comprising one or more enzymes, one or more dNTPs, and a buffer, wherein the buffer comprises one or more buffering reagents, one or more salts, and one or more crowding reagents; b) incubating the sample with the set of primers and the solution at a reaction temperature for a length of time; and c) producing single-stranded oligonucleotide amplicons having a sequence comprising at least the target region of the template nucleic acid molecule.
  2. 2 . The method of claim 1 , wherein the ratio of P1:P2:P3 in the set of primers is 1:(10-200):10.
  3. 3 . The method of claim 1 , wherein the target region is from 20 to 500 bases in length.
  4. 4 . The method of claim 1 , wherein the buffer further comprises a reducing agent.
  5. 5 . The method of claim 1 , wherein the one or more enzymes comprise a recombinase enzyme, a single strand binding protein, a strand displacing polymerase, a reverse transcriptase, or a combination thereof.
  6. 6 . The method of claim 5 , wherein the recombinase enzyme is RecA, Rad51, or RadA.
  7. 7 . The method of claim 5 , wherein the single-stranded binding protein is Escherichia coli single-stranded DNA binding protein (EcSSB) or T4 GP32.
  8. 8 . The method of claim 5 , wherein the strand displacing polymerase is Bacillus subtilis DNA polymerase I (Bsu), or mesophilic DNA polymerase.
  9. 9 . The method of claim 1 , wherein the one or more buffering reagents are tris(hydroxymethyl)aminomethane (Tris), phosphate buffered saline (PBS), or a combination thereof.
  10. 10 . The method of claim 1 , wherein the one or more salts comprise sodium chloride (NaCl), potassium chloride (KCl), magnesium chloride (MgCl 2 ), sodium acetate (CH 3 COONa), magnesium acetate (Mg(CH 3 COO) 2 ), monosodium phosphate (NaH 2 PO 4 ), disodium phosphate (Na 2 HPO 4 ), or a combination thereof.
  11. 11 . The method of claim 1 , wherein the one or more crowding reagents are polyvinylpyrrolidone (PVP), polyethylene glycol (PEG), polysucrose, Dextran, or a combination thereof.
  12. 12 . The method of claim 1 , wherein the reaction temperature ranges from 15° C. to 60° C.
  13. 13 . The method of claim 1 , wherein the length of time is from 5 to 60 minutes.
  14. 14 . The method of claim 1 , wherein the buffer has a pH ranging from 7.0-8.0.
  15. 15 . The method of claim 1 , further comprising detecting the target region.
  16. 16 . The method of claim 15 , wherein detection of the target region comprises introducing a genosensor probe.
  17. 17 . A method of real time asymmetric semi-nested isothermal nucleotide amplification (ANINA) for producing and quantifying single-stranded oligonucleotide amplicons comprising a target region of a template nucleic acid molecule, said method comprising: a) introducing to a sample comprising the template nucleic acid molecule: i) a set of primers comprising: A) a first primer (P1) comprising a first template nucleotide sequence, wherein the entire first template nucleotide sequence is complementary to a first complementary binding region (CSBR1), wherein said P1 is 5′ to the target region, and wherein said CSBR1 is on a strand opposite to the target region; B) a second primer (P2) comprising a second template nucleotide sequence, wherein the entire second template nucleotide sequence is complementary to a second complementary binding region (CSBR2), wherein P2 is (i) 5′ to the target region or (ii) 5′ to the target region and comprises a portion of the target region, wherein said CSBR2 is on a strand opposite to the target region, and wherein P1 is at least partially 5′ to P2; C) a third primer (P3) comprising a third template nucleotide sequence, wherein the entire third template nucleotide sequence is complementary to a target strand binding region (TSBR) of the target strand, wherein the TSBR comprises (i) at least a portion of the target region, (ii) a portion 3′ to the target region, or (iii) any combination of (i) and (ii), wherein the ratio of P1:P2:P3 in the set of primers is (1-10):(10-200):(1-20); and ii) a solution comprising one or more enzymes, one or more dNTPs, and a buffer, wherein the buffer comprises one or more buffering reagents, one or more salts, and one or more crowding reagents; b) incubating the sample with the set of primers and the solution at a reaction temperature for a length of time; and c) producing single-stranded oligonucleotide amplicons having a sequence comprising at least the target region of the nucleic acid molecule; wherein the method quantifies the single-stranded oligonucleotide amplicons produced when the single-stranded oligonucleotide amplicons bind to a reporter probe.
  18. 18 . The method of claim 17 , wherein the one or more enzymes comprises a recombinase enzyme, a strand displacing polymerase, a reverse transcriptase, or a combination thereof.
  19. 19 . The method of claim 18 , wherein the recombinase enzyme is RecA, Rad51, or RadA.
  20. 20 . The method of claim 18 , wherein the strand displacing polymerase is Bacillus subtilis DNA polymerase I (Bsu), Bst, or Klenow Fragment.

Description

CROSS-REFERENCES TO RELATED APPLICATIONS This application is a non-provisional and claims benefit of U.S. Provisional Application No. 63/171,761 filed Apr. 7, 2021 and U.S. Provisional Application No. 63/240,227 filed Sep. 2, 2021, the specification(s) of which is/are incorporated herein in their entirety by reference. This application is a non-provisional and claims benefit of U.S. Provisional Application No. 63/183,504 filed May 3, 2021, the specification of which is incorporated herein in their entirety by reference. FIELD OF THE INVENTION The present invention features an asymmetric semi-nested isothermal nucleotide amplification (ANINA) method, as well as compositions and systems, for the amplification of single-stranded oligonucleotides from a target sequence. BACKGROUND OF THE INVENTION Nucleic acid testing (NAT) biosensing technologies, such as those employing the use of fluorophores or redox sensors or other physicochemical detectors, are currently used in numerous applications for the detection of nucleic acids (NA) of a particular pathogen or mutated genes in an organism. NATs use the specificity and sensitivity afforded by nucleic acid base pairing to detect different NA sequences, sometimes differing by a single nucleotide. However, for a NAT to work effectively, single-stranded DNA and/or RNA need to be isolated for detection. This allows for a physicochemical detector (e.g., a genosensor probe) to hybridize to its complementary sequence, which should be a single-stranded DNA and/or RNA target. This can be achieved by fragmenting the genomic DNA/RNA or by amplifying the target sequence using nucleic acid amplification technologies (NAATs), which increases the number of target copies and the sensitivity of the detection. However, both of the aforementioned methods have shortcomings associated with them. First, fragmenting DNA in a controlled manner to a size between 25-100 bps is extremely difficult to achieve. Most mechanical and chemical breakdown methods give ˜1 kbps fragments which are not preferred as longer sequences are more difficult to denature and tend to form secondary structures, thereby decreasing the hybridization efficiency. Additionally, enzymatic methods usually use DNAses that have a propensity of depolymerizing DNA to fragments less than 25 bps. Even the commercially available DNAses, such as fragmentase (NEB), are difficult to control to give size-specific fragments. Lastly, the nucleic acid fragments need to be denatured, if they are double-stranded, to bind to the single-stranded probe, leading to competition between the probe and homologous strand which makes the process more complicated and inefficient. Furthermore, there is the potential for non-specific binding to the probe of other fragments. The most used NAAT is PCR which remains the gold standard of current diagnostics. However, PCR is difficult to conduct in a point-of-need setting, because of the requirement of specific cycling temperatures: for example, (i) a very high temperature (usually 95° C.) required for thermal melting of dsDNA/dsRNA (either the starting sequence or the amplicons after the first amplification cycle), (ii) annealing temperature (usually between 50-70° C.) for the primers to bind to, and (iii) the extension temperature for the corresponding amplification by the common polymerases (e.g., Taq (72° C.)). Additionally, PCR and real-time PCR are limited to the time required for each cycle, thereby increasing the total time of the reactions. Other isothermal NAATs, such as LAMP (loop-mediated isothermal amplification), HDA (helicase-dependent isothermal DNA amplification). SDA (strand displacement amplification), NASBA (nucleic acid sequence-based amplification), RCA (rolling circle amplification), can amplify DNA in a simpler setting by avoiding the need for a cycling method. However, all the aforementioned methods still require either thermal melting of the dsDNA or dsRNA, or incubation at a temperature higher than 50° C. or require the addition of finicky nucleases adding to the complexity of the process. Additionally, most NAATs can amplify if there is a contamination, and therefore nested amplification strategies are utilized to make the amplicon results more specific. Furthermore, most NAATs utilize a dual strand amplification strategy to increase the number of copies exponentially. However, this results in dsDNA amplicons, which still need to be denatured before being detected. While asymmetric amplification strategies can be used to amplify mostly single-stranded DNA, it heavily limits the amplification rate to a linear rate instead of the exponential rate of the dual-strand approach. Currently, an isothermal cost-effective nucleic acid amplification technology (NAAT) that specifically amplifies single-strand oligonucleotides is needed. Furthermore, a method that does not require thermal melting and can be done in a point-of-need setting would be ideal. BRIEF SUMMARY OF THE INVENTION It i