Systems and methods for predicting oligonucleotide melting temperature (TmS)

• US 7,715,989 B2
• Filed: 06/18/2002
• Issued: 05/11/2010
• Est. Priority Date: 04/03/1998
• Status: Expired due to Fees

• Current Assignee: ELITechGroup Inc. (ELITech Group SAS)
• Original Assignee: Elitech Holding BV (ELITech Group SAS)
• Inventors: Gall, Alexander A., Singer, legal representative, Singer, Michael J., Dempcy, Robert O., Vermeulen, Nicolaas M. J., Lokhov, Sergey G., Kutyavin, Igor V., Afonina, Irina A.
• Primary Examiner(s): Lin; Jerry
• Application Number:
  US10/176,972
• Publication Number:
  US 20030235822A1
• Time in Patent Office: 2,884 Days
• Field of Search: 435/6, 702/19, 702/20
• US Class Current: 702/20
• CPC Class Codes: C07B 2200/11 Compounds covalently bound to a solid support
• CPC Class Codes: C07H 19/06 Pyrimidine radicals
• CPC Class Codes: C07H 19/10 with the saccharide radical esterified by phosphoric or polyphosphoric acids
• CPC Class Codes: C07H 19/16 Purine radicals
• CPC Class Codes: C07H 19/20 with the saccharide radical esterified by phosphoric or polyphosphoric acids
• CPC Class Codes: C07H 19/23 Heterocyclic radicals containing two or more heterocyclic rings condensed among themselves or condensed with a common carbocyclic ring system, not provided for in groups C07H19/14 - C07H19/22
• CPC Class Codes: C07H 21/00 Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• CPC Class Codes: C12Q 1/6813 Hybridisation assays
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 1/6858 Allele-specific amplification
• CPC Class Codes: C12Q 2525/117 incorporating modified base
• CPC Class Codes: C12Q 2525/197 incorporating a spacer/coupling moiety
• CPC Class Codes: C12Q 2527/107 Temperature of melting, i.e. Tm
• CPC Class Codes: C12Q 2535/125 Allele specific primer extension
• CPC Class Codes: C12Q 2541/101 Selex
• CPC Class Codes: C12Q 2563/173 staining/intercalating agent, e.g. ethidium bromide
• CPC Class Codes: C12Q 2600/156 Polymorphic or mutational markers
• CPC Class Codes: C40B 40/00 Libraries per se, e.g. arrays, mixtures
• CPC Class Codes: G16B 25/00 ICT specially adapted for hybridisation; ICT specially adapted for gene or protein expression
• CPC Class Codes: G16B 30/00 ICT specially adapted for sequence analysis involving nucleotides or amino acids

Abstract

Software systems and methods for predicting the melting temperature (Tm) and other characteristics of oligonucleotides, including modified oligonucleotides. Modified oligonucleotides are provided containing bases selected from unsubstituted and 3-substituted pyrazolo[3,4-d]pyrimidines and 5-substituted pyrimidines, and optionally have attached minor groove binders and reporter groups.

Claims

1. A method of predicting the melting temperature (T_m) of an oligonucleotide sequence, comprising:

inputting into a computer system data representing a sequence of N bases of an identified oligonucleotide having N-1 neighboring base pairs;

calculating in the computer system a (T_m) of the identified oligonucleotide sequence using the nearest neighbor thermodynamic parameters for each of the N-1 neighboring base pairs in the identified sequence, wherein said identified sequence includes at least one modified base selected from the group consisting of unsubstituted and 3-substituted pyrazolo[3,4-d]pyrimidines and 5-substituted pyrimidines, each nearest neighbor thermodynamic parameter defining a thermodynamic contribution of two corresponding neighboring bases; and

providing an output to a display or a user using the processing module, wherein said output represents a T_m of the identified oligonucleotide sequence.

2. The method of claim 1, wherein inputting includes receiving a polynucleotide sequence from GenBank.

3. The method of claim 1, wherein inputting includes receiving a polynucleotide sequence and identifying one of a portion of the received sequence and a sequence complementary to a portion of the received sequence.

4. The method of claim 1, wherein said at least one modified base includes a member selected from the group consisting of a base attached to an amino acid and a locked nucleic acid sugar.

5. The method of claim 1, wherein said at least one modified base includes a member selected from the group consisting of PPA (4-amino-1H-pyrazolo [3,4-d]pyrimidine), PPG (6-amino-1H-pyrazolo[3,4-d]pyrimidin-4(5H)-one), PPPA (4-amino-3-(prop-1-ynyl)pyrazolo[3,4-d]pyrimidine), PPPG (6-amino-3-prop-1-ynyl-5-hydropyrazolo[3,4-d]pyrimidine-4-one), PU (5-prop-1-ynyl-1,3-dihydropyrimidine-2,4-dione), PC (6-amino-5-prop-1-ynyl-3-dihydropyrimidine-2-one), HOPU (5-(3-hydroxyprop-1-ynyl)-1,3-dihydropyrimidine-2,4-dione), HOBuU (5-(4-hydroxybut-1-ynyl)-1,3-dihydropyrimidine-2,4-dione), HOBuC (6-amino-5-(4-hydroxybut-1-ynyl)-3-hydropyrimidine-2-one), HOBuPPA (4-(4-aminopyrazolo[3,4-d]pyrimidin-3-yl)but-3-yn-1-ol), (NH₂)₂PPPA (3-prop-1-ynylpyrazolo[3,4-d]pyrimidine-4,6-diamino), (NH₂)₂PPPAOH (2-(4,6-diaminopyrazolo[3,4-d]pyrimidin-3-yl)ethyn-1-ol), (NH₂)₂BuPPAOH (4-(4,6-diaminopyrazolo[3,4-d]pyrimidin-3-yl)but-3-yn-1-ol), (NH₂)₂PPAI (4,6-diamino-3-iodopyrazolo[3,4-d]pyrimidine), (NH₂)₂PPABr (3-bromo-4,6-diaminopyrazolo[3,4-d]pyrimidine), (NH₂)₂PPACl (3-chloro-4,6-diaminopyrazolo[3,4-d]pyrimidine), PPAI (4-amino-3-(iodo)pyrazolo[3,4-d]pyrimidine), PPABr (3-bromo-4-aminopyrazolo[3,4-d]pyrimidine), PPAC1 (3-chloro-4-aminopyrazolo[3,4-d]pyrimidine) and HOBuPPG (6-amino-3-(4-hydroxybut-1-ynyl)pyrazolo[3,4-d]pyrimidin-4-ol).

6. The method of claim 1, wherein said identified oligonucleotide sequence has a minor groove binder (MGB) attached thereto.

7. The method of claim 6, wherein said MGB has a formula selected from the group consisting of:

8. The method of claim 1, further comprising:

retrieving said nearest neighbor thermodynamic parameters for each of the N-1 neighboring base pairs in the identified sequence from a memory.

20. The method of claim 1, wherein said identified oligonucleotide sequence has attached to it one or more of a minor groove binder (MGB), a fluorophore (Fl) and a quencher (Q).

21. The method of claim 7, where the minor groove binder is attached to the oligonucleotide via a quencher molecule.

22. The method of claim 1, wherein identifying includes receiving a polynucleotide sequence from a sequence source.

23. The method of claim 22, wherein the sequence source includes a GenBank server.

9. A computer readable storage medium including computer code for controlling a processor to calculate the melting temperature (T_m) of an oligonucleotide, the computer code including instructions that cause the processor to perform the following steps:

input into a processing module data representing a sequence of N bases of an identified oligonucleotide having N-1 neighboring base pairs;

retrieve nearest neighbor thermodynamic parameters for each of the N-1 neighboring base pairs in the identified sequence from a memory, wherein said identified sequence includes at least one modified base selected from the group consisting of unsubstituted and 3-substituted pyrazolo[3,4-d]pyrimidines and 5-substituted pyrimidines, each nearest neighbor thermodynamic parameter defining a thermodynamic contribution of two corresponding neighboring bases;

calculate a melting temperature (T_m) of the identified oligonucleotide sequence using the retrieved nearest neighbor thermodynamic parameters; and

provide an output to a display or a user using the processing module, wherein said output represents a T_m of the identified oligonucleotide sequence.

10. The computer-readable storage medium of claim 9, wherein said input includes a Genbank code, wherein said instructions to identify include instructions to automatically retrieve a polynucleotide sequence from Genbank over a network using said input Genbank code.

11. The computer-readable storage medium of claim 9, wherein said input includes a polynucleotide sequence.

12. The computer-readable storage medium of claim 9, wherein said instructions to identify includes instructions to identify one of a portion of a received polynucleotide sequence and a sequence complementary to a portion of the received sequence.

13. The computer-readable storage medium of claim 9, wherein said at least one modified base includes a member selected from the group consisting of a base attached to an amino acid and a locked nucleic acid sugar.

14. The computer-readable storage medium of claim 9, wherein said at least one modified base includes a member selected from the group consisting of PPA (4-amino-1H-pyrazolo[3,4-d]pyrimidine), PPG (6-amino-1H-pyrazolo[3,4-d]pyrimidin-4(5H)-one), PPPA (4-amino-3-(prop-1-ynyl)pyrazolo[3,4-d]pyrimidine), PPPG (6-amino-3-prop-1-ynyl-5-hydropyrazolo[3,4-d]pyrimidine-4-one), PU (5-prop-1-ynyl-1,3-dihydropyrimidine-2,4-dione), PC (6-amino-5-prop-1-ynyl-3-dihydropyrimidine-2-one), HOPU (5-(3-hydroxyprop-1-ynyl)-1,3-dihydropyrimidine-2,4-dione), HOBuU (5-(4-hydroxybut-1-ynyl)-1,3-dihydropyrimidine-2,4-dione), HOBuC (6-amino-5-(4-hydroxybut-1-ynyl)-3-hydropyrimidine-2-one), HOBuPPA (4-(4-aminopyrazolo[3,4-d]pyrimidin-3-yl)but-3-yn-1-ol), (NH₂)₂PPPA (3-prop-1-ynylpyrazolo[3,4-d]pyrimidine-4,6-diamino), (NH₂)₂PPPAOH (2-(4,6-diaminopyrazolo[3,4-d]pyrimidin-3-yl)ethyn-1-ol), (NH₂)₂BuPPAOH (4-(4,6-diaminopyrazolo[3,4-d]pyrimidin-3-yl)but-3-yn-1-ol), (NH₂)₂PPAI (4,6-diamino-3-iodopyrazolo [3,4-d]pyrimidine), (NH₂)₂PPABr (3-bromo-4,6-diaminopyrazolo[3,4-d]pyrimidine), (NH₂)₂PPACl (3-chloro-4,6-diaminopyrazolo[3,4-d]pyrimidine), PPAI (4-amino-3-(iodo)pyrazolo[3,4-d]pyrimidine), PPABr (3-bromo-4-aminopyrazolo[3,4-d]pyrimidine), PPACl (3-chloro-4-aminopyrazolo[3,4-d]pyrimidine) and HOBuPPG (6-amino-3-(4-hydroxybut-1-ynyl)pyrazolo[3,4-d]pyrimidin-4-ol).

15. The computer-readable storage medium of claim 9, wherein said identified oligonucleotide sequence has a minor groove binder (MGB) attached thereto.

16. The computer-readable storage medium of claim 15, wherein said MGB has a formula selected from the group consisting of:

24. The computer readable storage medium of claim 9, wherein the storage medium is selected from the group consisting of a floppy disk, CD and DVD.

17. A computer system for predicting the melting temperature (T_m) of an oligonucleotide sequence, comprising:

a memory unit for storing nearest neighbor thermodynamic parameters;

user interface means for identifying a nucleotide sequence having N bases and N-1 neighboring base pairs, wherein at least one of said N bases includes a modified base, wherein the means for identifying includes means for receiving a user entered nucleotide sequence and;

processing means, coupled to the memory unit, for retrieving nearest neighbor parameters for each of the N-1 neighboring base pairs in the identified sequence from the memory unit, for calculating a melting temperature (T_m) of the identified oligonucleotide sequence using the nearest neighbor thermodynamic parameters, and for providing an output to a display or a user, wherein said output represents a T_m of the identified oligonucleotide sequence, and wherein said identified sequence includes at least one modified base selected from the group consisting of unsubstituted and 3-substituted pyrazolo[3,4-d]pyrimidines and 5-substituted pyrimidines, each nearest neighbor thermodynamic parameter defining a thermodynamic contribution of two corresponding neighboring bases.

18. The system of claim 17, wherein the means for identifying includes means for receiving a GenBank GenInfo Identifier (GI) number specifying a nucleotide sequence stored at a GenBank server, the system further including means for retrieving the specified sequence from a GenBank server.

19. The system of claim 17, wherein the means for identifying includes means for receiving a polynucleotide sequence having N or greater bases and identifying one of a portion of the received sequence and a sequence complementary to a portion of the received sequence.