METHODS FOR STRUCTURAL DETERMINATION OF SELENIUM DERIVATIZED NUCLEIC ACID COMPLEXES

• US 20160279250A1
• Filed: 11/21/2014
• Published: 09/29/2016
• Est. Priority Date: 11/21/2013
• Status: Active Grant

• Current Assignee: Sena Research, Inc.
• Original Assignee: Sena Research, Inc.
• Inventors: HUANG, Zhen
• Granted Patent: US 10,441,637 B2
• Time in Patent Office: Days
• US Class Current: 1/1
• CPC Class Codes: A61K 38/465 acting on ester bonds (3.1), e.g. lipases, ribonucleases
• CPC Class Codes: A61K 47/61 the organic macromolecular compound being a polysaccharide or a derivative thereof
• CPC Class Codes: C07K 1/30 by precipitation
• CPC Class Codes: C12N 9/22 Ribonucleases RNAses, DNAses catalytic nucleic acids C12N15/113
• CPC Class Codes: C12N 9/96 Stabilising an enzyme by forming an adduct or a composition; Forming enzyme conjugates
• CPC Class Codes: C12Y 301/26004 Ribonuclease H (3.1.26.4)
• CPC Class Codes: G01N 33/5308 for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites
• CPC Class Codes: G16H 50/50 for simulation or modelling of medical disorders
• CPC Class Codes: G16Z 99/00 Subject matter not provided for in other main groups of this subclass
• CPC Class Codes: Y02A 90/10 Information and communication technologies [ICT] supporting adaptation to climate change, e.g. for weather forecasting or climate simulation

Abstract

Methods for crystallizing a molecule of interest, such as a polypeptide, in complex with nucleic acid, including contacting the molecule of interest with selenium-derivatized nucleic acid and crystallizing the molecule of interest/selenium-derivatized nucleic acid complex are provided. Methods for determining the X-ray crystal structure of molecule of interest/selenium-derivatized nucleic acid complexes are also provided. Typically, the method of X-ray crystal structural determination includes selenium single-wavelength anomalous phasing of the selenium-derivatized nucleic acid. In some embodiments the phases for the X-ray crystal structure of the molecule of interest are not provided from another crystal. Also disclosed are methods of affecting a biological process by administering a functional nucleic acid to a cell or a subject and/or by bringing into contact a nuclease and a functional nucleic acid, where the functional nucleic acid is selenium-derivatized nucleic acid.

Claims

1. A method of crystallizing a molecule of interest in complex with nucleic acid, comprising the steps of

(a) contacting the molecule of interest with selenium-derivatized nucleic acid;

and

(b) crystallizing the molecule of interest/selenium-derivatized nucleic acid complex.

2. The method of claim 1, further comprising the step of determining the X-ray crystal structure of the molecule of interest/selenium-derivatized nucleic acid complex.

3. The method of claim 2, wherein the X-ray crystal structural determination comprises selenium single-wavelength anomalous phasing of the selenium-derivatized nucleic acid.

4. The method of claim 2, wherein the resolution of diffraction of the X-ray crystallographic data obtained with the selenium-derivatized nucleic acid complex is greater than that of crystallographic data obtained in the absence of selenium-derivatized nucleic acid.

5. The method of claim 1, wherein the molecule of interest does not crystallize in the absence of selenium-derivatized nucleic acid.

6. The method of claim 2, wherein phases for the X-ray crystal structure of the molecule of interest are not provided from another crystal.

7. The method of claim 1, wherein the molecule of interest is a polypeptide.

8. The method of claim 1, wherein the molecule of interest is not a nucleic acid-binding protein.

17. The method of claim 1, wherein the selenium-derivatized nucleic acid is selenium-derivatized RNA.

18. The method of claim 1, wherein the selenium-derivatized nucleic acid binds to the molecule of interest with at least the same affinity as nucleic acid that is not selenium-derivatized.

19. The method of claim 1, wherein the selenium-derivatized nucleic acid binds the same region of the molecule of interest as nucleic acid that is not selenium-derivatized.

9. A method of determining the interaction between a molecule of interest and nucleic acid, comprising the steps of

(a) contacting the molecule of interest with a selenium-derivatized nucleic acid;

(b) crystallizing the molecule of interest/selenium-derivatized nucleic acid complex;

(c) determining the three dimensional X-ray crystal structure of the molecule of interest/selenium-derivatized nucleic acid complex, wherein the method of X-ray crystal structural determination comprises selenium single-wavelength anomalous phasing of the selenium-derivatized nucleic acid; and

(d) identifying the interfacing elements between the molecule of interest and selenium-derivatized nucleic acid in the three dimensional X-ray crystal structure of the molecule of interest/selenium-derivatized nucleic acid complex.

10. The method of claim 9, further comprising selecting a candidate molecule that interferes with the interaction between the molecule of interest and the nucleic acid, wherein the candidate molecule is selected by

(a) providing on a digital computer the three-dimensional X-ray crystal structure of the molecule of interest/selenium-derivatized nucleic acid complex;

and

(b) selecting a candidate molecule predicted to bind the nucleic acid interface identified in the polypeptide/selenium-derivatized nucleic acid complex structure.

11. The method of claim 9, further comprising designing a candidate molecule that binds to the molecule of interest, wherein the candidate molecule is designed by

(a) providing on a digital computer the three-dimensional crystal structure of the molecule of interest/selenium-derivatized nucleic acid complex;

and

(b) using software comprised by the digital computer to design a candidate molecule that is predicted to bind to the polypeptide.

12. The method of claim 11, further comprising

(a) synthesizing the candidate molecule;

and

(b) evaluating the candidate molecule for an ability to alter an activity of the molecule of interest.

16. The method of claim 12, wherein the molecule of interest is a polypeptide.

13. A method of selecting crystals containing selenium-derivatized nucleic acid, comprising the steps of

(a) growing crystals in a solution containing selenium-derivatized nucleic acid;

(b) selecting crystals that are colored yellow, indicating the presence of selenium-derivatized nucleic acid.

14. The method of claim 13, wherein the color of crystals containing selenium-derivatized nucleic acid is compared to the color of control crystals that do not contain selenium-derivatized nucleic acid.

15. The method of claim 13, wherein the selenium-derivatized nucleic acid is in complex with a molecule of interest.

20. A complex of a molecule of interest and a selenium-derivatized nucleic acid.

21. The complex of claim 20, wherein the molecule of interest is a polypeptide.

22. A method of affecting a biological process, the method comprising administering a functional nucleic acid to a cell or a subject, wherein the functional nucleic acid is selenium-derivatized nucleic acid.

23. The method of claim 22, wherein the functional nucleic acid is an aptamer, an antisense nucleic acid, an siRNA, or an shRNA.

24. The method of claim 22, wherein the functional nucleic acid binds to or affects a molecule of interest.

26. The method of claim 22, wherein the functional nucleic acid is a substrate or cofactor for a nuclease.

27. The method of claim 26, wherein the nuclease is an endonuclease, an exonuclease, a ribonuclease, a deoxyribonuclease, a ribozyme, a transcription activator-like effector nuclease (TALEN), a Zinc finger nuclease, an RNA-induced silencing complexes (RISC), an Argonaute, a Dicer, a CRISPR-associated (Cas) nuclease, a restriction enzyme, a meganuclease, an RNase A, an RNase H, an RNase I, an RNase III, an RNase L, an RNase P, an RNase T1, an RNase T2, an RNase U2, an RNase V, an RNase V1, an RNase PhyM, an RNase II, an RNase R, an RNase PH, an RNase D, an RNase T, a polynucleotide phosphorylase (RNPase), an oligoribonuclease, an exoribonuclease I, an exoribonuclease II, a micrococcal nuclease, an S1 nuclease, a P1 nuclease, a peptidyl transferase 23S rRNA, a Group I intron, a Group II intron, a GIR1 branching enzyme, a leadzyme, a hairpin ribozyme, a twister ribozyme, a hammerhead ribozyme, an HDV ribozyme, a mammalian CPEB3 ribozyme, a VS ribozyme, a glmS ribozyme, or a CoTC ribozyme.

28. The method of claim 26, wherein catalytic activity of the nuclease is increased in the presence of the selenium-derivatized nucleic acid compared to catalytic activity of the nuclease in the presence of a corresponding nucleic acid that is not selenium-derivatized.

29. The method of claim 26, wherein the cofactor is be a substrate-guiding sequence that directs a nuclease to cleave a substrate.

25. A method of affecting a biological process, the method comprising bringing into contact a nuclease and a functional nucleic acid, wherein the functional nucleic acid is selenium-derivatized nucleic acid.