Methods for structural determination of selenium derivatized nucleic acid complexes

• US 10,441,637 B2
• Filed: 11/21/2014
• Issued: 10/15/2019
• Est. Priority Date: 11/21/2013
• Status: Active Grant

• Current Assignee: Sena Research, Inc.
• Original Assignee: Sena Research, Inc.
• Inventors: Huang, Zhen
• Primary Examiner(s): Noakes, Suzanne M
• Application Number:
  US15/038,242
• Publication Number:
  US 20160279250A1
• Time in Patent Office: 1,789 Days
• Field of Search: None
• 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 for crystallographic analysis of a molecule of interest, comprising the steps of

(a) contacting the molecule of interest with selenium-derivatized nucleic acid to form a molecule of interest/selenium-derivatized nucleic acid complex,

wherein the selenium-derivatized nucleic acid comprises a selenium at the 6-position of guanosine;

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

(c) determining the X-ray crystal structure of the molecule of interest/selenium-derivatized nucleic acid complex, wherein the X-ray crystal structural determination comprises single-wavelength anomalous diffraction (SAD) phasing of the selenium-derivatized nucleic acid.

2. The method of claim 1, 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.

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

4. The method of claim 1, wherein phases for the X-ray crystal structure of the molecule of interest are not provided from another crystal.

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

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

7. The method of claim 1, further comprising

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.

8. The method of claim 7, 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.

9. The method of claim 7, 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.

10. The method of claim 9, 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.

11. The method of claim 10, wherein the molecule of interest is a polypeptide.

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

13. 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.

14. 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.