PNA-DNA oligomers and methods of use thereof

• US 20080131880A1
• Filed: 11/22/2006
• Published: 06/05/2008
• Est. Priority Date: 11/22/2006
• Status: Abandoned Application

• Current Assignee: Massachusetts Institute of Technology
• Original Assignee: Massachusetts Institute of Technology
• Inventors: Bortolin, Laura T., Rudzinski, Christina M., Stephens, Amanda L.
• Application Number:
  US11/604,014
• Publication Number:
  US 20080131880A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6823 Release of bound markers
• CPC Class Codes: C12Q 2521/319 Exonuclease
• CPC Class Codes: C12Q 2525/107 incorporating a peptide nucleic acid
• CPC Class Codes: C12Q 2525/197 incorporating a spacer/coupling moiety
• CPC Class Codes: C12Q 2565/30 Detection characterised by liberation or release of label

Abstract

Peptide nucleic acid (PNA)-deoxyribonucleic acid (DNA) oligomers and methods of using the PNA-DNA oligomers to detect and/or amplify a target nucleic acid in a sample are provided. The PNA-DNA oligomers of the invention are relatively insensitive to ionic concentration and many inhibitory proteins and, consequently, are particularly advantageous for direct use in environmental or challenging samples to detect nucleic acid, especially that of microorganisms, including food and water pathogens and/or bioterrorism agents. Methods are also provided for use of the PNA-DNA oligomers in applications including polymerase chain reaction, nucleic acid sequencing, mutation detection and as nucleic acid probes.

Claims

1. A PNA-DNA oligomer comprising:

a peptide nucleic acid (PNA) oligomer and a deoxyribonucleic acid (DNA) oligomer, wherein the PNA oligomer and DNA oligomer are covalently linked by a C₆ amino linker having the formula;

2. The PNA-DNA oligomer of claim 1, wherein the PNA is about 1 to about 7 bases.

3. A method for detecting the presence of a target nucleic acid in a sample comprising:

a) combining a peptide nucleic acid (PNA)-deoxyribonucleic acid (DNA) oligomer, a labeled probe, a DNA polymerase having exonuclease activity, unlabeled deoxyribonucleotide triphosphates (dNTPs) and said sample, thereby forming a combination;

b) maintaining said combination under conditions suitable for extending said PNA-DNA oligomer in the presence of the target nucleic acid wherein;

i) said PNA-DNA oligomer and labeled probed are annealed to said target nucleic acid and wherein said labeled probe anneals to the target nucleic acid downstream of the PNA-DNA oligomer; and

ii) said DNA polymerase extends said PNA-DNA oligomer annealed to the target nucleic acid in the presence of the unlabeled deoxyribonucleotide triphosphates (dNTPs), thereby forming a full-length unlabeled nucleic acid product, wherein the exonuclease activity of the DNA polymerase degrades the labeled probe annealed to the target nucleic acid during extension of the PNA-DNA oligomer annealed to the target nucleic acid, thereby resulting in emission of a detectable signal; and

c) analyzing said combination for emission of said detectable signal, wherein emission of the detectable signal indicates the presence of the target nucleic acid in the sample.

4. The method of claim 3, wherein the labeled probe is fluorescently labeled with a high energy dye on the 5′

end of the probe and a low energy dye on the 3′

end of the probe in a fluorescent resonance energy transfer (FRET) format.

5. The method of claim 4, wherein the labeled probe is at least about 17 bases long and anneals between about 1 to about 5 bases downstream of the PNA-DNA oligomer.

6. The method of claim 3, wherein the method further comprises amplifying said full-length unlabeled nucleic acid product.

7. The method of claim 3, wherein the PNA-DNA oligomer is comprised of a PNA oligomer and a DNA oligomer, wherein the PNA oligomer and DNA oligomer are covalently linked by a C₆ amino linker having the formula:

8. The method of claim 3, wherein the PNA-DNA oligomer has a purine content of less than about 60%, a guanine-cytosine (GC) content of between about 30% to about 80% and single nucleotide repeats of less than about 4 bases.

9. The method of claim 3, wherein said sample is selected from the group consisting of a biological sample, an environmental sample, a contaminated sample, a suboptimal sample, a substantially purified sample and a pristine sample.

10. The method of claim 9, wherein said biological sample is selected from the group consisting of muscous, saliva, urine, feces, whole blood, plasma, serum, cerebrospinal fluid, alveolar lavages, sweat, tears, a carcass sample, a nasal swab sample and combinations thereof.

11. The method of claim 9, wherein said environmental sample is selected from the group consisting of water, soil, air, sewage, food, crops, plant tissue, surface wipes, forensic samples and combinations thereof.

12. The method of claim 9, wherein said suboptimal sample is selected from the group consisting of a bacterial culture supernatant, a food sample, a sample with a high pH or a low pH, a sample with a high salt concentration or a low salt concentration, a sample with inhibitory proteins, a sample with heavy metals and combination thereof.

13. The method of claim 3, wherein emission of said detectable signal indicates the presence of a target nucleic acid of one or more microorganisms.

14. The method of claim 13, wherein said one or more microorganisms is a pathogen.

15. A method for detecting the presence of a target ribonucleic acid (RNA) in a sample comprising:

a) combining a first peptide nucleic acid (PNA)-deoxyribonucleic acid

(DNA) oligomer, a reverse transcriptase enzyme, unlabeled deoxyribonucleotide phosphates (dNTPs) and said sample, thereby forming a first combination;

b) maintaining said combination under conditions suitable for extending said first PNA-DNA oligomer in the presence of the target RNA wherein;

i) said first PNA-DNA oligomer anneals to said target RNA in said sample; and

ii) said reverse transcriptase enzyme extends said first PNA-DNA oligomer annealed to the target RNA in the presence of the unlabeled dNTPs, thereby forming a full-length unlabeled cDNA product;

c) combining a second PNA-DNA oligomer, a labeled probe, a DNA polymerase having exonuclease activity and the full-length unlabeled cDNA product, thereby forming a second combination;

d) maintaining said second combination under conditions suitable for extending said second PNA-DNA oligomer in the presence of the full-length unlabeled cDNA product wherein;

i) said second PNA-DNA oligomer and labeled probed are annealed to the full-length unlabeled cDNA product and wherein said labeled probe anneals to the full-length unlabeled cDNA product downstream of the second PNA-DNA oligomer; and

ii) said DNA polymerase extends said second PNA-DNA oligomer annealed to said full-length unlabeled cDNA product in the presence of the unlabeled dNTPs, thereby forming a full-length unlabeled DNA product, wherein the exonuclease activity of the DNA polymerase degrades the labeled probe annealed to the full-length unlabeled cDNA product during extension of the second PNA-DNA oligomer annealed to the full-length unlabeled cDNA product, thereby resulting in emission of a detectable signal; and

e) analyzing said second combination for emission of said detectable signal, wherein emission of said detectable signal indicates the presence of said target RNA in the sample.

16. A method for detecting the presence of a target nucleic acid in a sample comprising:

a) combining a peptide nucleic acid (PNA)-deoxyribonucleic acid (DNA) oligomer, a DNA polymerase, unlabeled deoxyribonucleotide triphosphates (dNTPs), and said sample, thereby forming a combination;

b) maintaining said combination under conditions suitable for extending said PNA-DNA oligomer in the presence of the target nucleic acid wherein;

i) said PNA-DNA oligomer is annealed to said target nucleic acid; and

ii) said DNA polymerase extends said PNA-DNA oligomer annealed to the target nucleic acid in the presence of the unlabeled dNTPs, thereby forming a full-length unlabeled target nucleic acid product;

c) amplifying said full-length unlabeled target nucleic acid product comprising;

i) denaturing the full-length unlabeled target nucleic acid product,

ii) maintaining the PNA-DNA oligomer, the DNA polymerase, a reverse complementary primer and the denatured full-length unlabeled target nucleic acid product under conditions suitable for extending the PNA-DNA oligomer and said reverse complementary primer in the presence of the denatured full-length unlabeled target nucleic acid product, wherein the PNA-DNA oligomer and the reverse complementary primer anneal to the denatured full-length unlabeled target nucleic acid product and wherein the DNA polymerase extends the PNA-DNA oligomer and the reverse complementary primer annealed to the denatured full-length unlabeled target nucleic acid product in the presence of unlabeled dNTPs, thereby forming full-length unlabeled target nucleic acid product, and

iii) repeating steps i) and ii) one or more times, thereby producing one or more full-length unlabeled target nucleic acid products; and

d) detecting said one or more full-length unlabeled target nucleic acid products,

wherein the presence of one or more full-length target nucleic acid products indicates the presence of said target nucleic acid in the sample.

17. The method of claim 16, wherein said one or more full-length unlabeled target nucleic acid products are detected by a DNA-intercalating agent or a dye that preferentially binds double-stranded DNA.

18. A method of amplifying a target nucleic acid comprising:

a) combining a peptide nucleic acid (PNA)-deoxyribonucleic acid (DNA) oligomer, a reverse complementary primer, a labeled probe, a DNA polymerase having exonuclease activity, unlabeled deoxyribonucleotide triphosphates (dNTPs) and said target nucleic acid, thereby forming a combination;

b) maintaining said combination under conditions suitable for extending said PNA-DNA oligomer in the presence of the target nucleic acid wherein;

i) said PNA-DNA oligomer and labeled probed are annealed to said target nucleic acid and wherein said labeled probe anneals to the target nucleic acid downstream of the PNA-DNA oligomer; and

ii) said DNA polymerase extends said PNA-DNA oligomer annealed to the target nucleic acid in the presence of the unlabeled dNTPs, thereby forming full-length unlabeled target nucleic acid product, wherein the exonuclease activity of the DNA polymerase degrades the labeled probe annealed to the target nucleic acid during extension of the PNA-DNA oligomer annealed to the target nucleic acid, thereby resulting in emission of a detectable signal;

c) denaturing the full-length unlabeled target nucleic acid product and;

d) repeating steps b) and c) one or more times, thereby forming one or more unlabeled target nucleic acid products and thereby amplifying the target nucleic acid.

19. The method of claim 18 wherein said target nucleic acid is denatured prior to the annealing of said PNA-DNA oligomer, said reverse complementary primer and said labeled probe to said target nucleic acid.

20. The method of claim 18 further comprising detecting the emission of said detectable signal, thereby detecting the amplification of the target nucleic acid in real time.

21. The method of claim 18, wherein the PNA-DNA oligomer is comprised of a PNA oligomer and a DNA oligomer, wherein the PNA oligomer and DNA oligomer are covalently linked by a C₆ amino linker having the formula:

22. The method of claim 18 wherein the reverse complementary primer is a second PNA-DNA oligomer.

23. A method of amplifying a target nucleic acid comprising:

a) combining a peptide nucleic acid (PNA)-deoxyribonucleic acid (DNA) oligomer, a reverse complementary primer, a DNA polymerase, unlabeled deoxyribonucleotide triphosphates (dNTPs) and said target nucleic acid, thereby forming a combination;

b) maintaining the PNA-DNA oligomer, the DNA polymerase, a reverse complementary primer and the target nucleic acid, under conditions suitable for extending the PNA-DNA oligomer and said reverse complementary primer in the presence of the target nucleic acid, wherein;

i) the PNA-DNA oligomer and the reverse complementary primer anneal to the target nucleic acid, and

ii) the DNA polymerase extends the PNA-DNA oligomer and the reverse complementary primer annealed to the target nucleic acid, in the presence of the unlabeled dNTPs, thereby forming full-length unlabeled target nucleic acid product;

c) denaturing the full-length unlabeled target nucleic acid product; and

d) repeating steps b) and c) one or more times, thereby forming one or more full-length unlabeled target nucleic acid products and thereby amplifying the target nucleic acid.

24. The method claim 23 wherein said target nucleic acid is denatured prior to the annealing of said PNA-DNA oligomer and said reverse complementary primer to said target nucleic acid.

25. The method of claim 23 wherein said PNA-DNA oligomer comprises a PNA oligomer and a DNA oligomer, wherein the PNA oligomer and DNA oligomer are covalently linked by a C₆ amino linker having the formula:

26. The method of claim 23 wherein the reverse complementary primer is a second PNA-DNA oligomer.

27. A method of probing a target nucleic acid comprising:

a) hybridizing to the target nucleic acid an unlabeled peptide nucleic acid (PNA)-deoxyribonucleic acid (DNA) oligomer comprising at least one PNA oligomer and at least one DNA oligomer, wherein the at least one PNA oligomer and at least one DNA oligomer are covalently linked by a linker selected from the group consisting of a C₆ amino linker having the formula;

28. The method of claim 27, wherein said hybridized unlabeled PNA-DNA oligomer is detected by the cyanine dye 3,3′

-diethylthiadicarbocyanine iodide (DiSc₂(5)).

29. The method of claim 27, wherein said target nucleic acid is selected from the group consisting of synthetic nucleic acid, genetically engineered nucleic acid, genomic DNA, chromosomal DNA, mitochondrial DNA, transposon DNA, plasmid DNA, synthetic, RNA, ribosomal RNA, viral RNA and combinations thereof.

30. The method of claim 27, wherein the method is for detecting one or more nucleic acid changes selected from the group consisting of a mutation of, amplification of, addition to and deletion in said target nucleic acid.

31. A method for designing a peptide nucleic acid (PNA)-deoxyribonucleic acid (DNA) oligomer that binds a target nucleic acid comprising:

a) obtaining the sequence of the target nucleic acid; and

b) determining a complementary PNA-DNA oligomer sequence for a region on said target nucleic acid, thereby identifying a potential PNA-DNA oligomer, wherein the potential PNA-DNA oligomer is accepted or rejected in a method comprising;

i) calculating the percent of guanine (G) and cytosine (C) nucleotides in the potential PNA-DNA oligomer, wherein if said percent of G and C nucleotides is between about 30% and about 80%, then the potential PNA-DNA oligomer is accepted;

ii) calculating the melting temperature of the potential PNA-DNA oligomer, wherein if the melting temperature is between about 54°

C. and about 64°

C., then the potential PNA-DNA oligomer is accepted;

iii) determining the number of contiguous adenine (A), contiguous thymine (T), contiguous guanine (G) and contiguous cytosine (C) nucleotides in the potential PNA-DNA oligomer, wherein if there are less than;

(a) four contiguous A nucleotides, (b) four contiguous T nucleotides, (c) three contiguous C nucleotides and (d) three contiguous G nucleotides, then the potential PNA-DNA oligomer is accepted;

iv) calculating the percent of adenine (A) and guanine (G) nucleotides in the PNA oligomer portion of the potential PNA-DNA oligomer, thereby determining the purine content of the potential PNA-DNA oligomer, wherein if said percent of A and G nucleotides is less than or equal to about 60%, then the potential PNA-DNA oligomer is accepted;

v) calculating the percent of guanine (G) and cytosine (C) nucleotides in the PNA oligomer portion of the potential PNA-DNA oligomer, wherein if said percent of G and C nucleotides is between about 30% and about 80%, then the potential PNA-DNA oligomer is accepted;

vi) calculating the melting temperature of the PNA oligomer portion of the potential PNA-DNA oligomer, wherein if the melting temperature is between about 9°

C. and about 15°

C., then the potential PNA-DNA oligomer is accepted, and

vii) determining the number of contiguous G and C nucleotides in the

PNA oligomer portion of the potential PNA-DNA oligomer, wherein if there are not three contiguous G or three contiguous C nucleotides, then the PNA-DNA oligomer is accepted.

32. The method of claim 31 wherein the method is performed by a computer.

33. The method of claim 31, wherein the PNA-DNA oligomer is selected from the group consisting of a forward primer and a reverse primer.

34. The method of claim 31 further comprising designing a probe, the method further comprising:

a) determining a probe-binding region on said target nucleic acid located between 1 and 5 nucleotides 3′

to the region said PNA-DNA oligomer binds said target nucleic acid;

b) determining a complementary nucleotide sequence for said probe-binding region, thereby identifying a potential probe, wherein the potential probe is accepted or rejected in a method comprising;

i) determining a complementary nucleotide sequence to said probe-binding region, thereby identifying a potential probe;

ii) calculating the percent of cytosine (C) and guanine (G) nucleotides in said potential probe, wherein if the percent of C and G nucleotides is between about 30% and about 80%, then the potential probe is accepted;

iii) calculating the melting temperature for said potential probe, wherein if the melting temperature is between about 65°

C. and about 85°

C. then the potential probe is accepted;

iv) determining the number of contiguous adenine (A), contiguous thymine (T), contiguous guanine (G) and contiguous cytosine (C) nucleotides in the potential probe, wherein if there are less than;

(a) four contiguous A nucleotides, (b) four contiguous T nucleotides, (c) three contiguous C nucleotides and (d) three contiguous G nucleotides, then the potential probe is accepted; and

v) identifying the first nucleotide base of the potential probe, wherein if the first nucleotide base is an adenine (A), thymine (T) or a cytosine (C), then the potential probe is accepted.

35. The method of claim 34 wherein the method is performed by a computer.

36. A computer program product comprising a computer useable medium including a computer readable program, wherein the computer readable program, when executed on a computer causes the computer to:

a) obtain the sequence of the target nucleic acid; and

b) determine a complementary peptide nucleic acid (PNA)-deoxyribonucleic acid (DNA) oligomer sequence for a region on said target nucleic acid, thereby identifying a potential PNA-DNA oligomer;

c) calculate the percent of guanine (G) and cytosine (C) nucleotides in the potential PNA-DNA oligomer, wherein if said percent of G and C nucleotides is between about 30% and about 80%, then the computer accepts the potential PNA-DNA oligomer;

d) calculate the melting temperature of the potential PNA-DNA oligomer, wherein if the melting temperature is between about 54°

C. and about 64°

C., then the computer accepts the potential PNA-DNA oligomer;

e) determine the number of contiguous adenine (A), contiguous thymine (T), contiguous guanine (G) and contiguous cytosine (C) nucleotides in the potential PNA-DNA oligomer, wherein if there are less than;

(a) four contiguous A nucleotides, (b) four contiguous T nucleotides, (c) three contiguous C nucleotides and (d) three contiguous G nucleotides, then the computer accepts the potential PNA-DNA oligomer;

f) calculate the percent of adenine (A) and guanine (G) nucleotides in the PNA oligomer portion of the potential PNA-DNA oligomer, thereby determining the purine content of the potential PNA-DNA oligomer, wherein if said percent of A and G nucleotides is less than or equal to about 60%, then the computer accepts the potential PNA-DNA oligomer;

g) calculate the percent of guanine (G) and cytosine (C) nucleotides in the PNA oligomer portion of the potential PNA-DNA oligomer, wherein if said percent of G and C nucleotides is between about 30% and about 80%, then the computer accepts the potential PNA-DNA oligomer;

h) calculate the melting temperature of the PNA oligomer portion of the potential PNA-DNA oligomer, wherein if the melting temperature is between about 9°

C. and about 15°

C., then the computer accepts the potential PNA-DNA oligomer; and

i) determine the number of contiguous G and C nucleotides in the PNA oligomer portion of the potential PNA-DNA oligomer, wherein if there are not three contiguous G or three contiguous C nucleotides, then the computer accepts the potential PNA-DNA oligomer.

37. A computer program product comprising a computer useable medium including a computer readable program, wherein the computer readable program, when executed on a computer causes the computer to:

a) determine a probe-binding region on said target nucleic acid located between 1 and 5 nucleotides 3′

to the region a peptide nucleic acid (PNA)-deoxyribonucleic acid (DNA) oligomer binds a target nucleic acid;

b) determine a complementary nucleotide sequence for said probe-binding region, thereby identifying a potential probe;

c) calculate the percent of cytosine (C) and guanine (G) nucleotides in said potential probe, wherein if the percent of C and G nucleotides is between about 30% and about 80%, then the computer accepts the potential probe;

d) calculate the melting temperature for said potential probe, wherein if the melting temperature is between about 65°

C. and about 85°

C. then the computer accepts the potential probe;

e) determine the number of contiguous adenine (A), contiguous thymine (T), contiguous guanine (G) and contiguous cytosine (C) nucleotides in the potential probe, wherein if there are less than;

(a) four contiguous A nucleotides, (b) four contiguous T nucleotides, (c) three contiguous C nucleotides and (d) three contiguous G nucleotides, then the computer accepts the potential probe and

f) identify the first nucleotide base of the potential probe, wherein if the first nucleotide base is an adenine (A), thymine (T) or a cytosine (C), then the computer accepts the potential probe.