Methods for detecting modification resistant nucleic acids

• US 9,284,547 B2
• Filed: 04/22/2013
• Issued: 03/15/2016
• Est. Priority Date: 11/03/2008
• Status: Active Grant

• Current Assignee: Regents of the University of California (University of California)
• Original Assignee: Regents of the University of California (University of California)
• Inventors: Weissman, Jonathan, Ingolia, Nicholas, Ghaemmaghami, Sina, Newman, John
• Primary Examiner(s): Ketter, Jim
• Application Number:
  US13/867,848
• Publication Number:
  US 20130288905A1
• Time in Patent Office: 1,058 Days
• Field of Search: None
• US Class Current: 1/1
• CPC Class Codes: C12N 15/1034 Isolating an individual clone by screening libraries
• CPC Class Codes: C12N 15/1041 Ribosome/Polysome display, e.g. SPERT, ARM
• CPC Class Codes: C12N 15/1093 General methods of preparing gene libraries, not provided for in other subgroups
• CPC Class Codes: C12Q 1/6809 Methods for determination or identification of nucleic acids involving differential detection
• CPC Class Codes: C40B 40/08 Libraries containing RNA or DNA which encodes proteins, e.g. gene libraries
• CPC Class Codes: C40B 50/06 Biochemical methods, e.g. using enzymes or whole viable microorganisms

Abstract

Methods are provided for, inter alia, detecting nucleic acid molecules resistant to degradation, such as a plurality of RNA molecules bound to a ribosome, using various technologies including deep sequencing.

Claims

1. A method for determining a protein synthesis rate, comprising:

(a) isolating a plurality of monosomes from a plurality of polysomes, wherein each of said plurality of polysomes comprises a ribosome bound to a portion of a translatable RNA molecule;

(b) sequencing each of said portions;

(c) determining a ribosome footprint density for each translatable RNA molecule from said sequencing; and

(d) determining a protein synthesis rate from the ribosomal footprint density.

2. The method of claim 1, wherein the determining of the ribosome footprint density comprises aligning each of said portions.

3. The method of claim 1, further comprising quantifying each of said portions.

4. The method of claim 1, wherein said isolating comprises contacting said plurality of polysomes with a degradant.

5. The method of claim 1, further comprising quantifying a relative amount of two or more of said portions.

6. The method of claim 2, further comprising determining a location of high ribosome footprint density within said translatable RNA molecule.

7. The method of claim 1, wherein said sequencing is determined by using a sequence by synthesis technique.

8. The method of claim 1, wherein said determining of said protein synthesis rate comprises determining expression of a plurality of protein coding genes expressed from said translatable RNA molecules.

9. The method of claim 8, wherein said protein coding genes are located on two or more different chromosomes.

10. The method of claim 4, further comprising removing ribosomal RNA prior to step (a).

11. The method of claim 1, further comprising amplifying the translatable RNA portion prior to sequencing.

12. A method for determining the protein synthesis rate for a plurality of protein coding genes, comprising:

(a) isolating a plurality of monosomes from a plurality of polysomes, wherein each of said plurality of polysomes comprises a plurality of ribosomes, wherein each of said plurality of ribosomes is bound to a portion of a mRNA molecule;

(b) aligning the sequence of each of said portions; and

(c) determining the ribosomal footprint density from said aligning, thereby determining the protein synthesis rate for a plurality of protein coding genes.

13. The method of claim 12, wherein said isolating comprises comprises contacting said plurality of polysomes with a degradant.

14. The method of claim 12, further comprising quantifying a relative amount of two or more of said portions.

15. The method of claim 12, further comprising removing ribosomal RNA prior to step (a).

16. The method of claim 12, further comprising determining a location of high ribosome footprint density within said mRNA molecule.

17. The method of claim 12, wherein said sequencing is determined by using a sequence by synthesis technique.

18. The method of claim 12, wherein said determining of said protein synthesis rate comprises determining expression of a plurality of protein coding genes expressed from said mRNA molecules.

19. The method of claim 18, wherein said protein coding genes are located on two or more different chromosomes.

20. The method of claim 12, further comprising amplifying the portion prior to sequencing.