Methods of use for thermostable RNA ligases

• US 20040058330A1
• Filed: 09/20/2002
• Published: 03/25/2004
• Est. Priority Date: 09/20/2002
• Status: Abandoned Application

• Current Assignee: Illumina Incorporated
• Original Assignee: Prokaria Itd.
• Inventors: Kristjansson, Jakob K., Fridjonsson, Olafur H., Skirnisdottir, Sigurlaug, Blondal, Thorarinn, Aevarsson, Arnthor, Hreggvidsson, Gudmundur O., Hjorleifsdottir, Sigridur
• Application Number:
  US10/251,667
• Publication Number:
  US 20040058330A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12N 15/1093 General methods of preparing gene libraries, not provided for in other subgroups
• CPC Class Codes: C12P 19/34 Polynucleotides, e.g. nucleic acids, oligoribonucleotides
• CPC Class Codes: C12Q 2521/501 Ligase

Abstract

The activity and functional properties of novel thermostable RNA ligases isolated from thermophilic bacteriophages are described. Also described are methods of using these thermostable RNA ligases for various applications including nucleotide labeling, oligonucleotide synthesis, gene synthesis, gene amplification and amplification of mRNA and synthesis of cDNA.

Claims

1. A method of ligating nucleotides or nucleotide analogs or nucleic acids containing nucleotides or nucleotide analogs, comprising

contacting nucleotides or nucleic acids with a thermostable RNA ligase, wherein

the ligase catalyzes a reaction of ligation of the nucleotides, nucleotide analogs or nucleic acids.

2. The method of claim 1 wherein the ligation is performed at a temperature of about 50°

C. to about 75°

C.

3. The method of claim 1 wherein the RNA ligase is derived from a thermostable bacteriophage.

4. The method of claim 1 wherein the nucleotides are RNA or DNA.

5. The method of claim 1 wherein the nucleotide analogs contain modified bases, modified sugars and/or modified phosphate groups.

6. The method of claim 1 wherein the nucleic acids are single-stranded RNA or DNA.

7. The method of claim 1 wherein the thermostable RNA ligase is an isolated RNA ligase selected from the group consisting of:

a) a RNA ligase obtained from a bacteriophage infecting a thermophilic bacteria;

b) a polypeptide comprising the amino acid sequence of SEQ ID NO;

2 or SEQ ID NO;

4;

c) a polypeptide encoded by a nucleic acid comprising the sequence of SEQ ID NO;

1 or SEQ ID NO;

3;

d) a polypeptide having at least 30% sequence identity with the amino acid sequence of SEQ ID NO;

2 or SEQ ID NO;

4; and

e) a fragment or derivative of a), b), c), or d).

8. A method of forming a phosphodiester bond between a 3′

hydroxyl nucleic acid acceptor and a 5′

phosphate nucleic acid donor, comprising;

a) contacting a 3′

hydroxyl nucleic acid acceptor; and

b) a 5′

phosphate nucleic acid donor with a thermostable RNA ligase, wherein a phophodiester bond is formed between the nucleic acids.

9. The method of claim 8, wherein the formation of the phosphodiester bond is formed at a temperature of about 50°

C. to about 75°

C.

10. A method of synthesizing an oligonucleotide polymer by repeating cycles of combining a primer oligonucleotide and a blocked oligonucleotide, comprising:

a) combining the primer oligonucleotide and an oligonucleotide blocked at the 3′

or 5′

end in the presence of a thermostable RNA ligase, thereby forming an extended primer with a blocked 3′

or 5′

end;

b) enzymatically removing the blocked phosphate group at the 3′

or 5′

end or enzymatically adding a phosphate group to the 5′

end of the extended primer; and

c) repeating a) and b) using the extended primer from b) as the primer for a) wherein an oligonucleotide polymer is formed.

11. The method of claim 10 wherein the formed oligonucleotide polymer comprises a gene or a part of a gene coding for a polypeptide.

12. The method of claim 10, wherein step (a) is performed at a temperature of about 50°

C. to about 75°

C.

13. A method for synthesizing a recombinant gene product, comprising:

a) providing an array of immobilized oligonucleotides comprising predetermined areas on a surface of a solid support, each area having immobilized thereon copies of an oligonucleotide;

b) hybridizing to said immobilized oligonucelotides first single stranded terminal regions of first nucleic acid strands to be ligated; and

c) ligating with a thermostable RNA ligase, the hybridized first end of a first nucleic acid and a second nucleic acid.

14. The method of claim 13 wherein the ligation is performed at a temperature of about 50°

C. to about 75°

C.

15. A method of detecting nucleic acids, comprising:

a) contacting a first probe, a second probe, a target nucleic acid sample and a thermostable RNA ligase, wherein the first probe and the second probe hybridize to the target nucleic acid sample such that the 5′

end of the first probe and the 3′

end of the second probe are adjacent and can be ligated, wherein at least the 5′

terminal nucleotide of the first probe and the 3′

terminal nucleotide of the second probe are deoxyribonucleotides; and

b) incubating the first probe, second probe, target sample and RNA ligase under conditions that promote hybridization of the probes to the target sequence and that promote ligation of the probes are ligated if the target sequence is present in the target sample.

16. The method of claim 15 wherein the ligation is performed at a temperature of about 50°

C. to about 75°

C.

17. A method of amplifying nucleic acids, comprising:

a) contacting a nucleic acid containing sample, wherein the sample comprises a pool of mRNase having a poly-A tail, with i) an oligonucleotide with a 5′

end and a 3′

end comprising an oligo-dT sequence at the 3′

end, a promoter sequence recognized by a RNA polymerase at the 5′

end and a transcription initiation region located between the oligo-dT sequence and the promoter sequence wherein the oligonucleotide is blocked at the 3′

end to prohibit extension, ii) an enzyme having reverse transcription activity which forms a double stranded promoter-primer sequence, iii) at least one enzyme having Rnase H activity, iv) an enzyme having RNA polymerase activity, and v) sufficient amounts of dNTPs and rNTPs;

b) maintaining the resulting reaction mixture under appropriate conditions for a sufficient amount of time for enzymatic activity, such that antisense RNA is formed in the absence of cDNA intermediates;

c) contacting the multiple copies of RNA with i) a thermostable RNA ligase, ii) a double stranded DNA complex comprising a double stranded DNA promoter sequence, wherein each strand contains a 5′

end and a 3′

end, the promoter sequence recognizable by a RNA polymerase wherein one strand of said complex has a stretch of RNA attached to the 5′

end thereof, iii) an enzyme having RNA polymerase activity, and iv) sufficient amounts of dNTPs and rNTPs; and

d) maintaining the resulting reaction mixture under appropriate conditions for a sufficient amount of time for the enzymatic processes to occur.

18. The method of claim 17 wherein the ligation is performed at a temperature of about 50°

C. to about 75°

C.

19. A method for selectively isolating total cell mRNA, comprising:

a) contacting a cell lysate comprising total cell mRNA and non-isolated ribosome with a thermostable RNA ligase under conditions wherein the ligase adds a 3′

label to the total cell mRNA to form modified total cell mRNA; and

b) isolating the modified total cell mRNA.

20. The method of claim 19 wherein the ligation is performed at a temperature of about 50°

C. to about 75°

C.

21. A method for synthesizing a repeat region of an oligonucleotide having a defined sequence, the repeat region including a repeated nucleotide that appears more than once in succession, comprising:

a) ligating an oligonucleotide primer to a 3′

-phosphate-blocked repeated nucleotide to form a 3′

-phosphate-blocked primer;

b) removing the 3′

-phosphate blocking group from the 3′

-phosphate-blocked primer using a 3′

-phosphatase enzyme, thereby making a deblocked primer without removing the 3′

-phosphate blocking group from unreacted 3′

-phosphate-blocked repeated nucleotide; and

c) repeating steps (a) and (b) using unreacted 3′

-phosphate-blocked repeated nucleotide from step (b) as the 3′

-phosphate-blocked repeated nucleotide of step (a) and the deblocked primer product of step (b) as the oligonucleotide primer of step (a) without prior separation of the unreacted 3′

-phosphate-blocked repeated nucleotide from the deblocked primer product, whereby the cycles are repeated to form an oligonucleotide having a defined sequence.

22. The method of claim 21 wherein the ligation is performed at a temperature of about 50°

C. to about 75°

C.

23. A method for insertion of a single-stranded RNA sequence into a cloning vector, comprising:

ligating in the presence of a thermostable RNA ligase, both termini of the single-stranded RNA sequence with a linear double-stranded cloning vector having single-stranded termini complementary to both termini of the single-stranded RNA sequence to form an annealed product, in which the complementary termini of the single-stranded RNA sequence is formed by attaching oligonucleotide linkers to the RNA sequence with a thermostable RNA ligase.

24. The method of claim 23 wherein the ligation is performed at a temperature of about 50°

C. to about 75°

C.

25. A method for forming a library of DNA sequences, comprising:

a) forming a library of target RNA fragments by contacting multiple copies of non-denatured target RNA sequences with a library of random oligonucleotides in the presence of a hydrolytic agent under conditions where a subgroup of the library of random oligonucleotides hybridize to the target RNA, whereupon the hydrolytic agent hydrolyzes the target RNA at a site near the 5′

end of each hybridized random oligonucleotide, and wherein the 3′

ends of each fragment contains the entire sequence to which a random oligonucleotide in the subgroup hybridized; and

b) forming a library of templates for primer extension from the library of target RNA fragments; and

forming a library of DNA sequences that are complementary to the target RNA fragments from the library of templates for primer extension by attaching a nucleic acid primer complement sequence to the 3′

end of each target RNA fragment with a thermostable RNA ligase.

26. The method of claim 25 wherein the ligation is performed at a temperature of about 50°

C. to about 75°

C.

27. A method for amplifying a 5′

end region of target mRNA, comprising;

a) dephoshorylating mRNA molecules with a free phosphate group at the 5′

-end;

b) removing the 5′

-cap on full-length mRNAs;

c) ligating linkers to the 5′

-end of decapped mRNA molecules using a thermostable RNA ligase;

d) synthesizing cDNA using reverse transcriptase; and

e) amplifying the cDNA by PCR.

28. The method of claim 27 wherein the ligation is performed at a temperature of about 50°

C. to about 75°

C.

29. A method of amplifying mRNA, comprising:

a) synthesizing cDNA, wherein the first strand of cDNA is synthesized by reverse transcription of target mRNA using a 5′

end-phosphorylated RT-primer that is specific for the target RNA wherein a hybrid DNA-RNA is generated;

b) degrading the hybrid DNA-RNA by treatment with RNase H to remove RNA;

c) circularizing the single-stranded cDNA to form concatemers with a thermostable RNA ligase; and

d) amplifying DNA by PCR using specific primers that are complementary to known sequences.

30. The method of claim 29 wherein the ligation is performed at a temperature of about 50°

C. to about 75°

C.

31. A method of amplifying nucleic acids with single primer, comprising:

a) hybridizing a mixture of nucleic acids with a degenerate or non-degenerate primer targeted to a single region in a target sequence;

b) synthesizing single-stranded DNA complementary to a region of said target sequence, said synthesis being primed by said degenerate or non-degenerate primer and catalyzed by a DNA polymerase or a reverse transcriptase, thereby performing linear amplification of said target sequence by repeated thermal cycling;

c) providing a second primer site to said single-stranded DNA by ligating an oligonucleotide to its 3′

end, wherein the ligation is catalyzed by a thermostable RNA;

d) amplifying the single-stranded DNA using a primer pair wherein a first primer comprises at least a part of the degerate or non-degenerate primer sequence, or wherein a first primer is targeted to a region downstream to the degenerate or non-degenerate primer sequence, and the second primer is complementary to the 3′

primer site of step (c).

32. The method of claim 31 wherein the single stranded DNA synthesized in step b) is purified.

33. The method of claim 31 wherein step c) is performed at a temperature of about 50°

C. to about 75°

C.

34. A method for sequencing oligonucleotides, comprising

a) contacting a target oligonucleotide with a thermostable RNA ligase under conditions wherein the ligase adds an auxilary oligonucleotide to the 3′

end of the target oligonucleotide; and

b) sequencing the oligonucleotide.

35. The method of claim 34 wherein the ligation is performed at a temperature of about 50°

C. to about 75°

C.