Methods for incorporating non-perfectly matched oligonucleotides into target-specific hybridization sequences

• US 20050064457A1
• Filed: 06/07/2004
• Published: 03/24/2005
• Est. Priority Date: 06/06/2003
• Status: Active Grant

• Current Assignee: Board of Regents of the University of Michigan (University of Michigan)
• Original Assignee: Board of Regents of the University of Michigan (University of Michigan)
• Inventors: Lee, Inhan
• Granted Patent: US 7,745,117 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6813 Hybridisation assays

Abstract

The present invention provides a method of designing target-specific hybridization sequences which include one or more mutations. The method of the invention comprises selecting a candidate target nucleotide sequence in a subject nucleotide sequence. A first complementary nucleotide sequence to the target nucleotide sequence is identified by applying the known bonding relationships of the nucleotides. Next, a second complementary nucleotide sequence having one or more mutations is constructed. The amount of the “target-second complementary nucleotide sequence” hybrid formed when the candidate target nucleotide sequence and second complementary nucleotide sequences are combined in the presence of interfering sequences is characterized and used to assess utility of the second complementary nucleotide sequence. The present invention also provides the target-specific hybridization sequences designed by the methods of the invention.

Claims

1. A method of designing a target-specific hybridization sequence, the method comprising:

a) selecting a candidate target nucleotide sequence in a subject nucleotide sequence;

b) identifying a first complementary nucleotide sequence to the target nucleotide sequence;

c) replacing nucleotides at each of selected positions P_n in the first complementary nucleotide sequence to form a second complementary nucleotide sequence, the second complementary nucleotide sequence having a mismatch at each of selected positions P_n when the second complementary nucleotide sequence is hybridized with the candidate target nucleotide sequence;

d) characterizing the amount of “

target nucleotide sequence-second complementary nucleotide sequence”

hybrid formed when the candidate target nucleotide sequence and the second complementary nucleotide sequences are combined in the presence of potentially interfering sequences; and

e) if the amount of “

target-second complementary nucleotide sequence”

hybrid is greater than a predetermined value, identifying the second complementary nucleotide sequence as a symbolic representation of target-specific hybridization sequence.

2. The method of claim 1 further comprising:

prior to step d, identifying one or more interfering nucleotide sequences, wherein the interfering nucleotide sequences have at least a predetermined number of sequential nucleotides in common with the second complementary nucleotide sequence;

3. The method of claim 2 wherein the interfering nucleotide sequences have at least 14 sequential nucleotides in common with the second complementary nucleotide sequence.

4. The method of claim 2 wherein the step of characterizing the amount of “

target-second complementary nucleotide sequence”

hybrid comprises;

calculating the differences between a hybridization function for a “

target-second complementary nucleotide sequence”

hybrid and an “

interfering sequences-second complementary nucleotide sequence”

hybrid, the hybridization function providing quantification of the amount of hybridization; and

step e comprises;

if the differences are such that the the amount of “

target-second complementary nucleotide sequence”

hybrid is greater than the predetermined value, identifying the second complementary nucleotide sequence as the target-specific hybridization sequence.

5. The method of claim 3 wherein the hybridization function is the melt temperature, the difference in Gibbs free energy, interaction energy, or the percent hybridization.

6. The method of claim 1 wherein the subject nucleotide sequence having a length equal to or greater than the candidate target nucleotide sequence

7. The method of claim 1 further comprising selecting an alternative target candidate and repeating steps b-g with the candidate target sequence replaced with the alternative candidate target sequence.

8. The method of claim 1 wherein the candidate target nucleotide sequence and the second complementary nucleotide sequence are not self complementary.

9. The method of claim 1 wherein the step of selecting the candidate target nucleotide sequence comprises:

selecting an initial starting position from an end in the subject nucleotide sequence;

identify a set of candidate target sequences beginning at a set of starting positions given by the formula Xo+nA that include a predetermined number of nucleotides, wherein Xo is the initial starting position, n is an integer greater than or equal to 0 and is limited so that no sequence extends past the end of the test sequence, and A is an offset number of nucleotides between adjacent members in the set of candidate sequences;

quantifying the amount of interfering sequences for each member in the set of candidate target sequences; and

arranging the set of candidate target sequences from a sequence with the least amount of interfering sequences to a sequence with the greatest amount of interfering sequences.

10. The method of claim 9 wherein steps a through e are recursively executed with a different selected member from the set of candidate target sequences until a predetermined number of target-specific hybridization sequences are identified, the selected member first being sequentially selected from the member of the set with the least amount of interfering sequences to the member with the greatest amount of interfering sequences.

11. The method of claim 10 wherein A is from 1 to 50 nucleotides in length, and each member in the set of candidate sequences has from about 15 to about 100 nucleotides.

12. The method of claim 11 wherein Xo is at a position from the first position to about the 1500^th position in the subject nucleotide sequence,

13. The method of claim 1 wherein the target nucleotide sequence is selected by:

choosing a sequence from the collection of nucleotide sequences;

identifying a nucleotide at a predetermined position from an end to the sequence; and

selecting as the candidate target nucleotide sequence a nucleotide sequence starting at the predetermined position that includes a predetermined number of nucleotides.

14. The method of claim 1 wherein the step of replacing nucleotides at each of selected positions P_n comprises:

determining a replacement position in the first complementary nucleotide sequence at which a G is located, wherein a predetermined number of nucleotides from each end of the subject nucleotide sequence are excluded from consideration when determining the replacement position; and

creating the second complementary nucleotide sequences by replacing the G at the replacement position with a nucleotide selected from the group consisting of T/U, C, and A.

15. The method of claim 1 wherein the step of replacing nucleotides at each of selected positions P_n comprises:

determining a replacement position in the first complementary nucleotide sequence at which a C is located, wherein a predetermined number of nucleotides from each end of the subject nucleotide sequence are excluded from consideration when determining the replacement position; and

creating the second complementary nucleotide sequences by replacing the C at the replacement position with a nucleotide selected from the group consisting of T/U, G, and A.

16. The method of claim 1 wherein the step of replacing nucleotides at each of selected positions P_n comprises:

determining a replacement position in the first complementary nucleotide sequence at which an A is located, wherein a predetermined number of nucleotides from each end of the subject nucleotide sequence are excluded from consideration when determining the replacement position; and

creating the second complementary nucleotide sequences by replacing the A at the replacement position with a nucleotide selected from the group consisting of T/U and C.

17. The method of claim 1 wherein the step of replacing nucleotides at each of selected positions P_n comprises:

determining a replacement position in the first complementary nucleotide sequence at which a T/U is located, wherein a predetermined number of nucleotides from each end of the subject nucleotide sequence are excluded from consideration when determining the replacement position; and

creating the second complementary nucleotide sequences by replacing the T/U at the replacement position with a nucleotide selected from the group consisting of A and C.

18. The method of claim 1 wherein the step of replacing nucleotides at each of selected positions P_n comprises:

determining a replacement position in the first complementary nucleotide sequence at which a G×

G fragment is located, wherein a predetermined number of nucleotides from each end of the subject nucleotide sequence are excluded from consideration when determining the replacement position and X is any nucleotide; and

creating the second complementary nucleotide sequences by replacing each G in the G×

G fragment independently with a nucleotide selected from the group consisting of A, T/U, and C.

19. The method of claim 1 wherein the step of replacing nucleotides at each of selected positions P_n comprises:

determining a replacement position in the first complementary nucleotide sequence at which a GG fragment is located, wherein a predetermined number of nucleotides from each end of the subject nucleotide sequence are excluded from consideration when determining the replacement position; and

creating the second complementary nucleotide sequences by replacing each G in the GG fragment independently with a nucleotide selected from the group consisting of A, T/U, and C.

20. The method of claim 1 wherein the step of replacing nucleotides at each of selected positions P_n comprises:

determining a replacement position in the first complementary nucleotide sequence at which a CG fragment is located, wherein a predetermined number of nucleotides from each end of the subject nucleotide sequence are excluded from consideration when determining the replacement position; and

creating the second complementary nucleotide sequences by replacing the C in the CG fragment with a nucleotide selected from the group consisting of A, T/U, and G and replacing the G in the CG fragment with a nucleotide selected from the group consisting of A, T/U, and C.

21. The method of claim 1 wherein the step of replacing nucleotides at each of selected positions P_n comprises:

determining a replacement position in the first complementary nucleotide sequence at which a CC fragment is located, wherein a predetermined number of nucleotides from each end of the subject nucleotide sequence are excluded from consideration when determining the replacement position; and

creating the second complementary nucleotide sequences by independently replacing each C in the CC fragment with a nucleotide selected from the group consisting of A, T/U, and G.

22. The method of claim 1 wherein the mismatch is a natural nucleotide mismatch.

23. The method of claim 1 wherein steps a-g are implemented on a microprocessor.

24. The method of claim 1 wherein the mismatch is at a position exclusive of a predetermined number of nucleotides at either end of the second complementary nucleotide sequence.

25. An assay comprising an actual sequence corresponding to the symbolic representation identified by the method of claim 1.

26. A drug identified by the assay of claim 25.

27. A drug comprising an actual sequence corresponding to the symbolic representation identified by the method of claim 1.

28. A single stranded or double stranded RNA sequence designed by the method of claim 1.

29. A method of designing a target-specific hybridization sequence, the method comprising:

a) selecting a candidate target nucleotide sequence in a subject nucleotide sequence;

b) identifying one or more interfering nucleotide sequences, wherein the interfering nucleotide sequences at least a predetermined number of sequential nucleotides in common with the candidate target nucleotide sequence;

c) identifying a first complementary nucleotide sequence to the target nucleotide sequence;

d) determining a set of replacement positions P_n in the first complementary nucleotide sequence by locating each position at which a G is located;

e) creating a set of second complementary nucleotide sequences by replacing each G at the replacement position with a nucleotide selected from the group consisting of T/U, C, and A, wherein each member of the set of second complementary nucleotide sequences has a single G replaced;

f) characterizing the amount of “

target-second complementary nucleotide sequence”

hybrid formed when the target and second complementary nucleotide sequences are combining the presence of potentially interfering sequences; and

g) if the amount of “

target-second complementary nucleotide sequence”

hybrid is greater than a predetermined value, identifying the second complementary nucleotide sequence as a symbolic representation of target-specific hybridization sequence.

30. The method of claim 29 further comprising:

prior to step f, identifying one or more interfering nucleotide sequences, wherein the interfering nucleotide sequences have at least a predetermined number of sequential nucleotides in common with the second complementary nucleotide sequence;

31. The method of claim 30 wherein the step of characterizing the amount of the “

target-second complementary nucleotide sequence”

hybrid comprises;

calculating the differences between a hybridization function for a “

target-second complementary nucleotide sequence”

hybrid and an “

interfering sequences-second complementary nucleotide sequence”

hybrid, the hybridization function providing quantification of the amount of hybridization; and

step g comprises;

if the differences are such that the the amount of the “

target-second complementary nucleotide sequence”

hybrid is greater than the predetermined value, identifying the second complementary nucleotide sequence as the target-specific hybridization sequence.

32. The method of claim 31 wherein the hybridization function is the melt temperature, the difference in Gibbs free energy, interaction energy, or the percent hybridization.

33. The method of claim 29 further comprising:

i) if the first complementary nucleotide sequence did not have a G or the differences were not greater than the predetermined value, determining a set of replacement positions P_n in the first complementary nucleotide sequence by locating each position at which a C is located;

j) creating the set of second complementary nucleotide sequences by replacing each C at the replacement position with a nucleotide selected from the group consisting of T/U, G, and A, wherein each member of the set of second complementary nucleotide sequences has a single C replaced;

k) characterizing the amount of “

target-second complementary nucleotide sequence”

hybrid formed when the target and second complementary nucleotide sequences are combining the presence of potentially interfering sequences; and

l) if the amount of “

target-second complementary nucleotide sequence”

hybrid is greater than a predetermined value, identifying the second complementary nucleotide sequence as the amount of “

target-second complementary nucleotide sequence”

hybrid.

34. The method of claim 33 further comprising:

m) if the first complementary nucleotide sequence did not have a C or the differences were not greater than the predetermined value, determining a set of replacement positions P_n in the first complementary nucleotide sequence by locating each position at which a A or T/U is located;

n) creating the set of second complementary nucleotide sequences by replacing each A at the replacement position with a nucleotide selected from the group consisting of T/U and C or by replacing each T/U at the replacement position with a nucleotide selected from the group consisting of A and C, wherein each member of the set of second complementary nucleotide sequences has a single C replaced.

o) characterizing the amount of “

target-second complementary nucleotide sequence”

hybrid formed when the target and second complementary nucleotide sequences are combining the presence of potentially interfering sequences; and

p) if the amount of “

target-second complementary nucleotide sequence”

hybrid is greater than a predetermined value, identifying the second complementary nucleotide sequence as the amount of “

target-second complementary nucleotide sequence”

hybrid.

35. The method of claim 29 wherein steps a-g are implemented on a microprocessor.

36. The method of claim 29 wherein the mismatch is at a position exclusive of a predetermined number of nucleotides at either end of the second complementary nucleotide sequence.

37. An assay comprising an actual sequence corresponding to the symbolic representation identified by the method of claim 29.

38. A drug identified by the assay of claim 37.

39. A drug comprising an actual sequence corresponding to the symbolic representation identified by the method of claim 29.

40. A single stranded or double stranded RNA sequence designed by the method of claim 29.

41. A target-specific hybridization sequence comprising:

a sequence of nucleotides that are complementary to target nucleotide sequences except for the occurrence of one or more mutations at selected positions P_n in which one or more nucleotides in the sequence of nucleotides are replaced by a natural nucleotide that renders the sequence of nucleotides non-complementary at positions P_n.

42. The target-specific hybridization sequence of claim 41 wherein the P_n are not within a predetermined number of nucleotides from an end of the sequence.

43. The target-specific hybridization sequence of claim 41 wherein the one or more mutations comprise a single G replaced in a perfectly-matched complementary nucleotide sequence with a nucleotide selected from the group consisting of C, T/U, and A, the perfectly-matched complementary nucleotide sequence being perfectly complementary to the target nucleotide sequence.

44. The target-specific hybridization sequence of claim 41 wherein the one or more mutations comprise a single C replaced in a perfectly-matched complementary nucleotide sequence with a nucleotide selected from the group consisting of G, T/U, and A, the perfectly-matched complementary nucleotide sequence being perfectly complementary to the target nucleotide sequence.

45. The target-specific hybridization sequence of claim 41 wherein the one or more mutations comprise a single A replaced in a perfectly-matched complementary nucleotide sequence with a nucleotide selected from the group consisting of C and T/U, the perfectly-matched complementary nucleotide sequence being perfectly complementary to the target nucleotide sequence.

46. The target-specific hybridization sequence of claim 41 wherein a single T/U is replaced in a perfectly-matched complementary nucleotide sequence with a nucleotide selected from the group consisting of C and A, the perfectly-matched complementary nucleotide sequence being perfectly complementary to the target nucleotide sequence.

47. The target-specific hybridization sequence of claim 41 wherein the one or more mutations comprise a fragment formed by individually replacing each G in a fragment G×

G present in a perfectly-matched complementary nucleotide sequence with a nucleotide selected from the group consisting of A, C and T/U and wherein X is any nucleotide, the perfectly-matched complementary nucleotide sequence being perfectly complementary to the target nucleotide sequence.

48. The target-specific hybridization sequence of claim 41 wherein the one or more mutations comprise a replacement fragment formed by individually replacing each G in a fragment GG present in a perfectly-matched complementary nucleotide sequence with a nucleotide selected from the group consisting of A, C and T/U, the perfectly-matched complementary nucleotide sequence being perfectly complementary to the target nucleotide sequence.

49. A computer readable medium having instructions thereon that perform the following steps for designing a target-specific hybridization sequence:

a) selecting a candidate target nucleotide sequence in a subject nucleotide sequence;

b) identifying a first complementary nucleotide sequence to the target nucleotide sequence;

c) replacing nucleotides at each of selected positions P_n in the first complementary nucleotide sequence to form a second complementary nucleotide sequence, the second complementary nucleotide sequence having a mismatch at each of selected positions P_n when the second complementary nucleotide sequence is hybridized with the candidate target nucleotide sequence;

d) characterizing the amount of “

target-second complementary nucleotide sequence”

hybrid formed when the target and second complementary nucleotide sequences are combining the presence of potentially interfering sequences; and

e) if the amount of “

target-second complementary nucleotide sequence”

hybrid is greater than a predetermined value, identifying the second complementary nucleotide sequence as a symbolic representation of target-specific hybridization sequence.