Immobilized RNPs for sequence-specific nucleic acid capture and digital detection

Patent metadata

Field	Value
Publication number	US-11905552-B2
Application number	US-201815998353-A
Country	US
Kind code	B2
Filing date	Aug 6, 2018
Priority date	Aug 4, 2017
Publication date	Feb 20, 2024
Grant date	Feb 20, 2024

Abstract

A digital biosensor for assaying a target nucleic acid and methods of using the biosensor for digitally detecting the target nucleic acid are disclosed wherein the biosensor includes a substrate having a substrate surface and at least two electrodes, a linker molecule having a first moiety conjugated to the substrate surface, a ribonucleoprotein (RNP) conjugated to a second moiety of the linker molecule, and a guide ribonucleic acid (gRNA) having a first sequence capable of binding to the inactive RNP and a second sequence capable of binding to the target nucleic acid.

First claim

What is claimed is:

1. A biosensor for assaying a target nucleic acid, the biosensor comprising:
a substrate comprising a substrate surface and at least two electrodes for passing current through the substrate, wherein the substrate surface comprises graphene;
a pyrenebutanoic acid (PBA) linker molecule comprising a pyrene ring moiety and a carboxylate moiety, wherein the linker molecule is conjugated to the substrate surface via non-covalent interaction between the pyrene ring moiety and the substrate surface;
a ribonucleoprotein (RNP) conjugated to the carboxylate moiety of the linker molecule via covalent bonding, wherein the RNP comprises Cas9 protein, wherein the linker molecule is joined to the Recombination UV C (RuvC) domain of the Cas9 protein, and wherein the RNP is immobilized to the substrate surface via the linker molecule; and
a guide ribonucleic acid (gRNA) forming a portion of the RNP and comprising a first sequence capable of binding to a protein portion of the RNP and a second sequence capable of binding to the target nucleic acid,
wherein conductivity of the substrate surface is modulated upon binding of the target nucleic acid to the gRNA, thereby enabling a detectable current change for sensing the target nucleic acid.

2. The biosensor of claim 1 wherein the RNP is active or inactive.

3. The biosensor of claim 1 , wherein the substrate surface further comprises silicon, germanene, graphene nanoribbons (GNR), bilayer graphene (BLG), phosphorene, stanine, graphene oxide, reduced graphene, fluorographene, MoS 2 , gold, or carbon.

4. The biosensor of claim 1 , wherein the substrate comprises a transistor comprising the at least two electrodes and the at least two electrodes comprise a source electrode, a drain electrode, and a gate electrode.

5. The biosensor of claim 4 , wherein the transistor is a field-effect transistor (FET).

6. The biosensor of claim 1 , wherein the pyrene ring moiety of the linker molecule is conjugated to the graphene of the substrate surface.

7. The biosensor of claim 1 , further comprising an electronic controller capable of measuring a change in current on the substrate surface.

8. The biosensor of claim 1 , wherein biosensor is configured such that the RNP and the gRNA forming the portion of the RNP remain attached to the substrate surface while modulating conductivity of the substrate surface upon binding of target nucleic acid to the gRNA.

9. A biosensor for assaying a target nucleic acid, the biosensor comprising:
a substrate comprising a substrate surface and at least two electrodes for passing current through the substrate, wherein the substrate surface comprises graphene;
a pyrenebutanoic acid (PBA) linker molecule comprising a pyrene ring moiety and a carboxylate moiety, wherein the linker molecule is conjugated to the substrate surface via non-covalent interaction between the pyrene ring moiety and the substrate surface; and
an immobilized RNP-gRNA probe that includes
a ribonucleoprotein (RNP) conjugated to the carboxylate moiety of the linker molecule via covalent bonding, wherein the RNP comprises inactive Cas9 protein, wherein the linker molecule is joined to the Recombination UV C (RuvC) domain of the inactive Cas9 protein, and wherein the RNP is immobilized to the substrate surface via the linker molecule, and
a guide ribonucleic acid (gRNA) comprising a first sequence bound to the RNP and a second sequence capable of binding to the target nucleic acid,

wherein conductivity of the substrate surface is modulated upon binding of target nucleic acid to the gRNA to thereby enable sensing of the target nucleic acid while the RNP-gRNA probe remains attached to the substrate surface.

10. A biosensor for assaying a target nucleic acid, the biosensor comprising:
a substrate comprising a substrate surface and at least two electrodes for passing current through the substrate, wherein the substrate surface comprises graphene;
a pyrenebutanoic acid (PBA) linker molecule comprising a pyrene ring moiety and a carboxylate moiety, wherein the linker molecule is conjugated to the substrate surface via interaction between the pyrene ring moiety and the substrate surface; and
an RNP-gRNA probe immobilized to the substrate surface via the linker molecule, the RNP-gRNA probe including
a ribonucleoprotein (RNP) comprising Cas9 protein coupled to a second moiety of the linker molecule via a carbodiimide bond, wherein the carbodiimide bond joins the linker molecule at the Recombination UV C (RuvC) domain of the Cas9 protein, and
a guide ribonucleic acid (gRNA) comprising a first sequence bound to the RNP and a second sequence capable of binding to the target nucleic acid,

wherein conductivity of the substrate surface is modulated upon binding of target nucleic acid to the gRNA to thereby enable sensing of the target nucleic acid while the RNP-gRNA probe remains attached to the substrate surface.

11. The biosensor of claim 9 , wherein the substrate comprises a transistor comprising the at least two electrodes and the at least two electrodes comprise a source electrode, a drain electrode, and a gate electrode.

12. The biosensor of claim 11 , wherein the transistor is a field-effect transistor (FET).

13. The biosensor of claim 9 , further comprising an electronic controller capable of measuring a change in current on the substrate surface.

14. The biosensor of claim 10 , wherein the substrate comprises a transistor comprising the at least two electrodes and the at least two electrodes comprise a source electrode, a drain electrode, and a gate electrode.

15. The biosensor of claim 14 , wherein the transistor is a field-effect transistor (FET).

16. The biosensor of claim 10 , further comprising an electronic controller capable of measuring a change in current on the substrate surface.

Assignees

• Keck Graduate Inst
• Keck Graduate Inst Of Applied Life Sciences
• Univ California

Inventors

• Aran Kiana
• Deboer Tara
• Conboy Irina

Classifications

• C12Q1/6825 Primary

  Nucleic acid detection involving sensors · CPC title

• C12N9/22

  Ribonucleases {[RNase]; Deoxyribonucleases [DNase]} · CPC title

• C12Q1/6806

  Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence) · CPC title

• C12Q1/6811

  Selection methods for production or design of target specific oligonucleotides or binding molecules · CPC title

• G01N27/3272

  Test elements therefor, i.e. disposable laminated substrates with electrodes, reagent and channels (optical biosensors G01N33/52) · CPC title