TM leveling methods
Methods for designing an oligonucleotide sequence having a selected duplex stability are provided wherein the oligonucleotide portion comprises at least one modified base selected from universal bases, unsubstituted and 3-substituted pyrazolo[3,4-d]pyrimidines and 5-substituted pyrimidines, and optionally having attached minor groove binders and reporter groups.
1. A method for designing a preferred oligonucleotide sequence having a selected duplex stability comprising:
a) providing an oligonucleotide sequence having N bases and N−1 neighboring base pairs,
b) modifying the oligonucleotide sequence to provide modified oligonucleotide sequences wherein said modified oligonucleotide sequences comprise at least one modified base selected from the group consisting of unsubstituted pyrazolo[3,4-d]pyrimidines, 3-substituted pyrazolo[3,4-d]pyrimidines, and 5-substituted pyrimidines;
c) calculating the duplex stabilities of said modified oligonucleotide sequences using an algorithm applying a nearest-neighbor model for duplex formation thermodynamics for each of the N−1 neighboring base pairs, each nearest neighbor thermodynamic parameter defining a thermodynamic contribution of two corresponding neighboring bases;
d) selecting a preferred oligonucleotide sequence from the modified oligonucleotide sequences having a selected duplex stability; and
e) creating an oligonucleotide comprising the preferred oligonucleotide sequence.
2. A method for designing a preferred oligonucleotide sequence having a selected duplex stability comprising:
a) providing an oligonucleotide sequence having N bases and N−1 neighboring base pairs,
b) modifying the oligonucleotide sequence to provide modified oligonucleotide sequences wherein said modified oligonucleotide sequences comprises comprise at least one modified base selected from the group consisting of unsubstituted pyrazolo[3,4-d]pyrimidines, 3-substituted pyrazolo[3,4-d]pyrimidines, and 5-substituted pyrimidines, and a minor groove binder;
c) calculating a melting temperature (T m ) of said modified oligonucleotide sequences using an algorithm applying nearest neighbor thermodynamic parameters for each of the N−1 neighboring base pairs, each nearest neighbor thermodynamic parameter defining a thermodynamic contribution of two corresponding neighboring bases;
d) selecting a preferred oligonucleotide sequence from the modified oligonucleotide sequences having the selected melting temperature; and
e) creating an oligonucleotide comprising the preferred oligonucleotide sequence.
3. The method of any one of claim 1 or 2 , wherein said oligonucleotide sequence is derived from a database source.
4. The method of claim 3 , wherein said database source is GENBANK.
5. The method of any one of claim 1 or 2 , wherein said at least one modified base is a member selected from the group consisting of a base attached to an amino acid, a polyamide nucleic acid (PNA), and a locked nucleic acid sugar.
6. The method of claim 5 , wherein said modified base is attached to PNA.
7. The method of claim 5 , wherein said modified base is attached to a locked nucleic acid sugar.
8. The method of any one of claim 1 or 2 , wherein said oligonucleotide has an enhanced ability of mismatch discrimination, in comparison to unmodified nucleotides.
9. The method of any one of claims 1 or 2 , wherein said at least one modified base is a member selected from the group consisting of PPA, PPG, PPPA, PPPG, PU, PC, HOPU, HOBuU, HOBuC, (NH 2 ) 2 PPPA, (NH 2 ) 2 PPPAOH, (NH 2 ) 2 BuPPAOH, (NH 2 ) 2 PPAI, and HOBuPPG.
10. The method of claim 1 , wherein said oligonucleotide has attached to it one or more members selected from the group consisting of a minor groove binder, a fluorophore, and a quencher.
11. The method of claim 10 , wherein said oligonucleotide has a minor groove binder attached thereto.
12. The method of claim 2 or 11 , wherein said minor groove binder has a formula selected from the group consisting of:
wherein
the subscript m is an integer of from 2 to 5;
the subscript r is an integer of from 2 to 10; and
each R a and R b is independently a linking group to said modified oligonucleotide, H, OR c , NR c R d , COOR c , or —CONR c R d wherein each R c and R d is selected from the group consisting of H, (C 1 -C 12 )heteroalkyl, (C 2 -C 12 )heteroalkenyl, (C 2 -C 12 )heteroalkynyl, (C 1 -C 12 )alkyl, (C 2 -C 12 )alkenyl, (C 2 -C 12 )alkynyl, aryl(C 1 -C 12 )alkyl, and aryl.
13. The method of claim 11 , wherein said minor groove binder is attached to the oligonucleotide via a quencher molecule.
14. The method of any one of claim 1 or 2 , wherein said algorithm predicts the melting temperature (T m ) of said oligonucleotide with an accuracy of about +/−2° C.
15. The method of any one of claim 1 or 2 , wherein said method is applied to establish appropriate conditions for hybridization, renaturation, mapping variations of base compositions of sequences, or determination of sequence complexity and divergence.
16. The method of any one of claim 1 or 2 , wherein said oligonucleotide is a capture probe in an array.
17. The method of claim 6 , wherein said oligonucleotide has an enhanced ability of mismatch discrimination, in comparison to unmodified nucleotides.
18. The method of claim 7 , wherein said oligonucleotide has an enhanced ability of mismatch discrimination, in comparison to unmodified nucleotides.
19. The method of claim 1 , wherein the oligonucleotide sequence comprises a universal base and wherein the modified oligonucleotide sequences comprise the universal base and at least one modified base selected from the group consisting of unsubstituted pyrazolo[3,4-d]pyrimidines, 3-substituted pyrazolo[3,4-d]pyrimidines, and 5-substituted pyrimidines.
20. The method of claim 2 , wherein the oligonucleotide sequence comprises a universal base and wherein the modified oligonucleotide sequences comprise the universal base and at least one modified base selected from the group consisting of unsubstituted pyrazolo[3,4-d]pyrimidines, 3-substituted pyrazolo[3,4-d]pyrimidines, and 5-substituted pyrimidines.