Methods for detecting modification resistant nucleic acids

• US 8,486,865 B2
• Filed: 11/03/2009
• Issued: 07/16/2013
• 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: Ghaemmaghami, Sina, Weissman, Jonathan, Ingolia, Nicholas, Newman, John
• Primary Examiner(s): MUMMERT, STEPHANIE KANE
• Application Number:
  US12/611,817
• Publication Number:
  US 20100120625A1
• Time in Patent Office: 1,351 Days
• Field of Search: None
• US Class Current: 506/7
• 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 detecting a plurality of mRNA molecules bound to at least one ribosome, said method comprising:

(i) contacting said plurality of mRNA molecules with an enzymatic degradant or a chemical degradant thereby forming a plurality of RNA fragments, wherein each RNA fragment comprises an RNA portion protected from said enzymatic degradant or said chemical degradant by a ribosome to which the RNA portion is bound;

(ii) amplifying said RNA fragments comprising said RNA portions to form a detectable number of amplified nucleic acid fragments; and

(iii) detecting said detectable number of amplified nucleic acid fragments, thereby detecting an amount of two or more of said RNA portions and correlating said amount of two or more of said RNA portions to a rate of translation of one or more of said plurality of mRNA molecules.

2. The method of claim 1, wherein said amount of two or more RNA portions are correlated to an amount of ribosomal activity on one or more of said plurality of mRNA molecules.

3. The method of claim 1, wherein said amount of two or more RNA portions are correlated to an amount of protein synthesis from one or more of said plurality of mRNA molecules.

4. The method of claim 1, wherein said amount of two or more RNA portions are correlated to locations for boundaries of translated sequences.

8. The method of claim 1, wherein said amplifying and detecting comprises:

(a) contacting said plurality of RNA fragments with a DNA polymerase and a DNA polymerase primer thereby forming a plurality of linear DNA molecules;

(b) contacting said plurality of linear DNA molecules with a ligase thereby forming a plurality of circularized DNA molecules;

(c) amplifying said plurality of circularized DNA thereby forming a detectable number of amplified DNA molecules; and

(d) detecting said detectable number of amplified DNA molecules thereby detecting said plurality of mRNA molecules bound to at least one ribosome.

9. The method according to claim 1, wherein said detecting comprises contacting said amplified nucleic acid fragments with a microarray.

10. The method according to claim 1, wherein said amplified nucleic acid fragments comprise a detectable label.

11. The method according to claim 9, wherein said microarray comprises a nucleic acid at least 90% complementary to at least one of the amplified DNA molecules.

12. The method according to claim 1, further comprising isolating the mRNA molecules from a cell.

13. The method of claim 12, wherein the plurality of mRNA molecules comprise at least 90% of the mRNA sequence species from the cell.

14. The method according to claim 1, wherein said plurality of mRNA molecules comprise at least 1,000 different sequence species.

15. The method according to claim 1, wherein each of the RNA fragments are partially sequenced using a sequencing-by-synthesis technique to determine the nucleotide sequences of the RNA portions.

27. The method according to claim 12, wherein said mRNA molecules are each bound to at least one ribosome prior to said isolating.

5. A method for detecting a plurality of mRNA molecules bound to at least one ribosome, said method comprising:

(i) contacting said plurality of mRNA molecules with an enzymatic degradant or a chemical degradant thereby forming a plurality of RNA fragments, wherein each RNA fragment comprises an RNA portion protected from said enzymatic degradant or said chemical degradant by a ribosome to which the RNA portion is bound;

(ii) amplifying said RNA fragments to form a detectable number of amplified nucleic acid fragments; and

(iii) detecting said detectable number of amplified nucleic acid fragments, thereby detecting said plurality of mRNA molecules bound to at least one ribosome,

wherein said amplifying and detecting comprises;

(a) contacting said plurality of RNA fragments with a DNA polymerase and a DNA polymerase primer thereby forming a plurality of linear DNA molecules;

(b) contacting said plurality of linear DNA molecules with a ligase thereby forming a plurality of circularized DNA molecules;

(c) amplifying said plurality of circularized DNA thereby forming a detectable number of amplified DNA molecules; and

(d) detecting said detectable number of amplified DNA molecules thereby detecting said plurality of mRNA molecules bound to at least one ribosome.

6. A method for detecting a plurality of mRNA molecules bound to at least one ribosome, said method comprising:

(i) contacting said plurality of mRNA molecules with an enzymatic degradant or a chemical degradant thereby forming a plurality of RNA fragments, wherein each RNA fragment comprises an RNA portion protected from said enzymatic degradant or said chemical degradant by a ribosome to which the RNA portion is bound;

(ii) amplifying said RNA fragments to form a detectable number of amplified nucleic acid fragments; and

(iii) detecting said detectable number of amplified nucleic acid fragments, thereby detecting said plurality of mRNA molecules bound to at least one ribosome,

wherein said plurality of mRNA molecules comprise at least 1,000 different sequence species.

7. A method for determining translation activity for a plurality of mRNA molecules, comprising:

(i) treating a plurality of RNA-ribosome complexes, wherein each RNA-ribosome complex comprises an mRNA molecule having an RNA portion that is bound to a ribosome, under conditions sufficient produce a plurality of RNA fragments, wherein each RNA fragment comprises an RNA portion, and wherein each RNA fragment comprises a nucleotide sequence that is longer than the nucleotide sequence of said RNA portion;

(ii) sequencing the plurality of RNA fragments to determine the nucleotide sequences of the RNA portions; and

(iii) comparing the nucleotide sequences of the plurality of RNA portions to the nucleotide sequences of the mRNA molecules, thereby determining translation activity for the plurality of mRNA molecules,

wherein an amount of two or more RNA portions are correlated to a rate of translation of one or more mRNA molecules.

16. A method for detecting an RNA molecule bound to at least one ribosome, said method comprising:

(i) contacting said RNA molecule with an enzymatic degradant or chemical degradant thereby forming an RNA fragment, wherein the RNA fragment comprises an RNA portion protected from said enzymatic degradant or said chemical degradant by a ribosome to which the RNA portion is bound;

(ii) contacting said RNA fragment with a DNA polymerase and a DNA polymerase primer thereby forming a linear DNA;

(iii) contacting said linear DNA with a ligase thereby forming a circularized DNA;

(iv) amplifying said circularized DNA thereby forming a detectable number of amplified DNA molecules; and

(v) detecting said detectable number of amplified DNA molecules thereby detecting said RNA molecule bound to at least one ribosome.

17. A method for determining translation activity for a plurality of mRNA molecules, comprising:

(i) treating a plurality of RNA-ribosome complexes, wherein each RNA-ribosome complex comprises an mRNA molecule having an RNA portion that is bound to a ribosome, under conditions sufficient produce a plurality of RNA fragments, wherein each RNA fragment comprises an RNA portion, and wherein each RNA fragment comprises a nucleotide sequence that is longer than the nucleotide sequence of said RNA portion;

(ii) sequencing the plurality of RNA fragments to determine the nucleotide sequences of the RNA portions; and

(iii) comparing the nucleotide sequences of the plurality of RNA portions to the nucleotide sequences of the mRNA molecules, thereby determining translation activity for the plurality of mRNA molecules.

18. The method of claim 17, wherein the plurality of RNA-ribosome complexes is isolated from a cell and comprises at least 90% of the mRNA species from the cell.

19. The method of claim 17, further comprising isolating the mRNA molecules from a cell.

20. The method of claim 17, further comprising isolating the RNA-ribosome complexes from a cell.

21. The method of claim 17, wherein the plurality of RNAs comprises at least 1,000 different sequence species.

22. The method of claim 17, further comprising quantifying the relative amounts of two or more of the RNA portions in the RNA-ribosome complexes.

23. The method of claim 17, wherein each of the RNA fragments are partially sequenced using a sequencing-by-synthesis technique to determine the nucleotide sequences of the RNA portions.

24. The method of claim 17, wherein the translation activity comprises a rate of translation for one or more RNA in the plurality of RNA-ribosome complexes.

25. The method of claim 17, wherein the translation activity comprises the locations for boundaries of translated sequences.

26. The method of claim 17, wherein the translation activity comprises the reading frame being translated.

28. A method for detecting a plurality of RNA molecules bound to at least one ribosome, said method comprising:

(i) contacting said plurality of RNA molecules with an enzymatic degradant or a chemical degradant thereby forming a plurality of RNA fragments, wherein each RNA fragment comprises an RNA portion protected from said enzymatic degradant or said chemical degradant by a ribosome to which the RNA portion is bound;

(ii) amplifying said RNA fragments to form a detectable number of amplified nucleic acid fragments; and

(iii) detecting said detectable number of amplified nucleic acid fragments, thereby detecting said plurality of RNA molecules bound to at least one ribosome;

wherein said amplifying and detecting comprises;

(a) contacting said plurality of RNA fragments with a DNA polymerase and a DNA polymerase primer thereby forming a plurality of linear DNA molecules;

(b) contacting said plurality of linear DNA molecules with a ligase thereby forming a plurality of circularized DNA molecules;

(c) amplifying said plurality of circularized DNA thereby forming a detectable number of amplified DNA molecules; and

(d) detecting said detectable number of amplified DNA molecules thereby detecting said plurality of RNA molecules bound to at least one ribosome.