Bioreactive allosteric polynucleotides

• US 6,630,306 B1
• Filed: 05/04/2001
• Issued: 10/07/2003
• Est. Priority Date: 12/19/1996
• Status: Expired due to Fees

• Current Assignee: Yale University
• Original Assignee: Yale University
• Inventors: Breaker, Ronald R.
• Primary Examiner(s): ZITOMER, STEPHANIE W
• Application Number:
  US09/849,069
• Time in Patent Office: 886 Days
• Field of Search: 435/6, 435/287.2, 435/196, 536/23.1, 436/501
• US Class Current: 506/14
• CPC Class Codes: C07K 2319/00 Fusion polypeptide
• CPC Class Codes: C12N 15/101 by chromatography, e.g. electrophoresis, ion-exchange, reverse phase
• CPC Class Codes: C12N 15/113 Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing when used in plants C12N15/8218
• CPC Class Codes: C12N 2310/111 spanning the whole gene, or a large part of it
• CPC Class Codes: C12N 2310/12 catalytic nucleic acids, e.g. ribozymes
• CPC Class Codes: C12N 2310/121 Hammerhead
• CPC Class Codes: C12N 2310/322 2'-R Modification
• CPC Class Codes: C12Q 1/6811 Selection methods for production or design of target specific oligonucleotides or binding molecules
• CPC Class Codes: C12Q 1/6825 Nucleic acid detection involving sensors
• CPC Class Codes: C12Q 2521/337 Ribozyme
• CPC Class Codes: C12Q 2525/205 Aptamer

Abstract

Polynucleotides having allosteric properties that modify a function or configuration of the polynucleotide with a chemical effector and/or physical signal are employed primarily as biosensors and/or enzymes for diagostic and catalytic purposes. In some preferred embodiments, the polynucleotides are DNA enzymes that are used in solution/suspension or attached to a solid support as biosensors to detect the presence or absence of a compound, its concentration, or physical change in a sample by observation of self-catalysis. Chemical effectors include organic compounds such as amino acids, amino acid derivatives, peptides, nucleosides, nucleotides, steroids, and mixtures of these with each other and with metal ions, cellular metabolites or blood components obtained from biological samples, steroids, pharmaceuticals, pesticides, herbicides, food toxins, and the like. Physical signals include radiation, temperature changes, and combinations thereof.

Claims

1. A DNA polynucleotide comprising an allosteric site and an enzyme domain spatially distinct from said allosteric site, wherein reversible interaction of a chemical effector with the allosteric site on the DNA polynucleotide reversibly alters the cleavage function or configuration of the DNA polynucleotide, wherein the chemical effector is a metal ion or a small molecule having a molecular weight of 300 Daltons or less.

2. The DNA polynucleotide according to claim 1 wherein the function or configuration of the DNA polynucleotide is altered in less than 60 minutes after interaction with the chemical effector.

6. The DNA polynucleotide according to claim 1 or 3 wherein the chemical effector is selected from the group consisting of amino acids, amino acid derivatives, peptides, nucleosides, nucleotides, and steroids.

7. A biosensor comprising the DNA polynucleotide according to claim 1 or 3.

12. The biosensor according to claim 7, wherein the DNA polynucleotide is attached to a solid support.

3. A DNA polynucleotide comprising an allosteric site and an enzyme domain spatially distinct from said allosteric site, wherein the rate of catalysis of the enzyme domain is reversibly modulated by interaction with a chemical effector, wherein the chemical effector is a metal ion or a small molecule having a molecular weight of 300 Daltons or less.

4. The DNA polynucleotide according to claim 3 wherein an observable change in the rate of catalysis of the enzyme domain occurs in less than 6 minutes of interaction with the chemical effector.

5. The DNA polynucleotide according to claim 4 wherein the observable change occurs in less than 1 minute.

11. The DNA polynucleotide according to claim 3 wherein the rate of catalysis of the enzyme is measured by observing enzyme self-cleavage or substrate cleavage.

8. A method for detecting the presence or absence of a compound or its concentration in a sample comprising contacting the sample with a DNA polynucleotide comprising an allosteric site and an enzyme domain spatially distinct from said allosteric site, wherein reversible interaction of the compound with the allosteric site alters the cleavage function or configuration of the polynucleotide relative to that of a control sample, and observing said alteration in the function or configuration of the DNA polynucleotide, wherein the chemical effector is a metal ion or small molecule having a molecular weight of 300 Daltons or less, and further wherein an alteration in function or configuration of the DNA polynucleotide indicates the presence or absence of a compound or its concentration in the sample.

9. The method according to claim 8 wherein the presence or absence of a compound or its concentration is detected by observation of an alteration in the cleavage function of the polynucleotide.

10. The method according to claim 8 wherein the chemical effector is selected from the group consisting of amino acids, amino acid derivatives, peptides, nucleosides, nucleotides, and steroids.

13. An RNA polynucleotide comprising an allosteric site and an enzyme domain spatially distinct from said allosteric site, having three stem components, stem I, stem II and stem III, wherein stem I and stem III are polynucleotide sequences which together form the enzyme domain and stem II is a polynucleotide sequence which forms the allosteric site, wherein interaction of a chemical effector with the allosteric site reversibly alters the cleavage function or configuration of the polynucleotide, further wherein the chemical effector is a metal ion or a small molecule having a molecular weight of 300 Daltons or less.

14. The polynucleotide according to claim 13 wherein the cleavage function or configuration of the polynucleotide is altered in less than 60 minutes after interaction with the chemical effector.

19. A biosensor comprising the polynucleotide according to claim 13 or 15.

20. A method for detecting the presence or absence of a compound or its concentration in a sample comprising contacting the sample with a polynucleotide according to claim 13 or 15, such that reversible interaction of the compound with the allosteric site alters the cleavage function or configuration of the polynucleotide relative to that of a control sample, and observing said alteration in the cleavage function or configuration of the polynucleotide, wherein the compound is a chemical effector that is a metal ion or small molecule having a molecular weight of 300 Daltons or less and further wherein an alteration in function or configuration of the polynucleotide indicates the presence or absence of a compound or its concentration in the sample.

21. The method according to claim 20 wherein the presence or absence of a compound or its concentration is detected by observation of an alteration in the cleavage function of the polynucleotide.

22. The method according to claim 20 wherein the compound is a chemical effector that is selected from the group consisting of amino acids, amino acid derivatives, peptides, nucleosides, nucleotides, and steroids.

24. A biosensor according to claim 19, wherein the polynucleotide is attached to a solid support.

15. An RNA polynucleotide comprising an allosteric site and an enzyme domain spatially distinct from said allosteric site, having three stem components, stem I, stem II and stem III, wherein stem I and stem III are polynucleotide sequences which together form the enzyme domain and stem II is a polynucleotide sequence which forms the allosteric site, wherein interaction of a chemical effector with the allosteric site reversibly modulates the rate of catalysis of the polynucleotide, further wherein the chemical effector is a metal ion or a small molecule having a molecular weight of 300 Daltons or less.

16. The polynucleotide according to claim 15 wherein an observable change in the rate of catalysis of the polynucleotide occurs in less than 6 minutes of interaction with the chemical effector.

17. The polynucleotide according to claim 16 wherein the observable change occurs in less than 1 minute.

18. The polynucleotide according to claim 15 wherein the chemical effector is selected from the group consisting of amino acids, amino acid derivatives, peptides, nucleosides, nucleotides, and steroids.

23. A polynucleotide according to claim 15 wherein the rate of catalysis of the enzyme is measured by observing enzyme self-cleavage or substrate cleavage.