Photoactive compounds and methods for biomolecule detection and sequencing

• US 10,538,808 B2
• Filed: 05/29/2018
• Issued: 01/21/2020
• Est. Priority Date: 05/26/2017
• Status: Active Grant

• Current Assignee: VIBRANT HOLDINGS, LLC
• Original Assignee: VIBRANT HOLDINGS, LLC
• Inventors: Rajasekaran, John J.
• Primary Examiner(s): Bhat, Narayan K
• Application Number:
  US15/991,706
• Publication Number:
  US 20190194745A1
• Time in Patent Office: 602 Days
• CPC Class Codes: C12Q 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 1/6876 Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
• CPC Class Codes: C12Q 2523/319 Photocleavage, photolysis, photoactivation
• CPC Class Codes: C12Q 2527/119 pH
• CPC Class Codes: C12Q 2535/101 Sanger sequencing method, i.e. oligonucleotide sequencing using primer elongation and dideoxynucleotides as chain terminators
• CPC Class Codes: C12Q 2565/607 being a sensor, e.g. electrode
• CPC Class Codes: C40B 30/04 by measuring the ability to specifically bind a target molecule, e.g. antibody-antigen binding, receptor-ligand binding

Abstract

Disclosed herein are compositions, probes, devices, and processes useful for detecting specific reactions and binding interactions with biological molecules. In certain embodiments, methods of binding one or more biomolecules to a solid support are disclosed. Methods of generating site-specific sequences for one or more biomolecules from a solid support are also disclosed. Biological complexes generated by these methods are also disclosed.

Claims

1. A method of determining a sequence of a target polynucleotide, comprising:

providing an array comprising a plurality of wells, wherein said wells comprise a target polynucleotide to be sequenced bound to a surface of said well, and wherein said plurality of wells each comprise a sensor for detecting an electronic signal from said wells;

performing a sequencing reaction comprising performing at least one cycle, each cycle comprising;

contacting said wells with a solution comprising reagents for performing a polymerase extension reaction, said solution comprising a modified nucleotide comprising a photoactive group and a removable blocking group;

exposing said well to conditions to promote incorporation of one of said modified nucleotides at the 3′

end of a primer or probe hybridized to said single polynucleotide;

washing said well to remove unbound modified nucleotides;

exposing said well to a wavelength of light to induce said photoactive group to generate and acid or a base, thereby generating a detectable change in ion concentration;

detecting the change in ion concentration with said sensor; and

if another cycle of the sequencing reaction is to be performed, removing said removable blocking group from said incorporated nucleotide.

2. The method of claim 1, wherein said electronic signal is specific to the identity of the base of the modified nucleotide added to the primer at each cycle.

3. The method of claim 1, wherein said electronic signal represents the pH of a solution in said well.

4. The method of claim 1, wherein said electronic signal is analyzed to determine a sequence of the target polynucleotide.

5. The method of claim 1, wherein said sensor is an ion-sensitive field effect transistor.

6. The method of claim 1, wherein said modified nucleotides comprise a nucleotide according to Formula I:

7. The method of claim 1, wherein said modified nucleotides comprise a nucleotide according to Formula II:

8. The method of claim 1, wherein said photoactive group is a photoacid generator or a photobase generator.

9. The method of claim 1, wherein said modified nucleotides comprise a nucleotide selected from the group consisting of:

10. The method of claim 1, wherein said removable blocking group is a reversible terminator.

11. The method of claim 1, wherein the photoactive group is photocleavable.

12. The method of claim 1, wherein the photoactive group is a photoacid or photobase generator.

13. The method of claim 1, wherein said set of modified nucleotides comprises only one of the group consisting of:

nucleotides comprising adenine, nucleotides comprising guanine, nucleotides comprising thymine, or nucleotides comprising cytosine.

14. The method of claim 1, wherein said set of modified nucleotides comprises nucleotides comprising adenine, guanine, cytosine, and thymine or uracil.

15. The method of claim 1, wherein said solution comprises a plurality of random primers.

16. The method of claim 1, wherein said reagents for performing a polymerase extension reaction comprise a primer capable of hybridizing to said single polynucleotide.

17. The method of claim 1, wherein, if another cycle is to be performed, the method further includes neutralizing the solution in the wells.

18. The method of claim 1, wherein said plurality of wells each comprise only a single target polynucleotide.

19. The method of claim 1, wherein said plurality of wells each comprise a clonal population of a target polynucleotide.

20. The method of claim 1, comprising performing 2 or more of said cycles, 5 or more of said cycles, 10 or more of said cycles, 20 or more of said cycles, or 50 or more cycles of said cycles.

21. A method of detecting a sequence identity of a target polynucleotide, comprising:

providing a substrate an immobilized target polynucleotide hybridized to a primer or probe;

contacting said immobilized target polynucleotide with a solution comprising reagents for performing a polymerase extension reaction, said solution comprising a set of modified nucleotides comprising a photoactive group and a blocking group;

exposing said substrate to conditions to promote incorporation of one of said modified nucleotides at the 3′

end of said primer or probe;

washing said substrate to remove unbound modified nucleotides;

exposing said immobilized target polynucleotide to a wavelength of light to induce said photoactive group to generate an acid or a base, thereby generating a detectable change in ion concentration in a solution surrounding said immobilized target polynucleotide if said modified nucleotide is incorporated into said target polynucleotide;

detecting said change in ion concentration; and

determining a sequence identity of said target polynucleotide from said detected change in ion concentration.

22. A method of detecting a target biomolecule, comprising:

providing probe capable of binding specifically to a target biomolecule, wherein said probe is bound to a photoacid generator or a photobase generator;

contacting a sample suspected of comprising said target biomolecule with said probe;

removing unbound probes from said sample;

exposing said sample to an wavelength of light capable of activating said photoacid generator or said photobase generator, such that said probe, if bound to said target biomolecule, releases an acid of a base upon exposure to said wavelength of light; and

detecting a concentration of ions in the sample, thereby identifying the presence or absence of said target analyte based on a change of said concentration of ion.