California Institute of Technology

Molecular Instruments, Inc.

Niles A Pierce

Robert Dirks

Peng Yin

Scott E Fraser

Lisa Hochrein

Harry M T Choi

Marianne Bronner

Shashank Gandhi

Harry Ming Tak Choi

Heyun Li

Zhewei Chen

Mikhail H Hanewich-Hollatz

Long Cai

Joseph Zadeh

Chun Hao R Chen

Aneesh Acharya

Jeffrey Robert Vieregg

Mike C Liu

Naeem S Husain

Brian R Wolfe

Harry MT Choi

The present disclosure relates to methods and compositions involving HCR reactions that involve initiators that are split into two or more parts. Effective HCR is dependent upon two or more of these split initiators being brought into proximity (e.g., via binding events mediated by a target) such that a full initiator is formed that is capable of triggering HCR signal amplification.

Fractional initiator hybridization chain reaction

Programmable guide RNAs (gRNAs) play a central role in the CRISPR revolution sweeping biology and medicine by directing the function of a Cas protein effector to a target gene of choice. To achieve programmable control over regulatory scope, the activity of a conditional guide RNA (cgRNA) depends on the presence or absence of an RNA trigger, allowing for cell-selective regulation of CRISPR/Cas function. Unlike a standard gRNA, a cgRNA is programmable at multiple levels, with the target-binding sequence controlling the target of Cas activity (edit, silence, induce, or bind a gene of choice) and the trigger-binding sequence controlling the scope of Cas activity. cgRNA mechanisms that are allosteric allow for independent design of the target and trigger sequences, providing the flexibility to select the regulatory target and scope independently. Disclosed herein are allosteric cgRNA mechanisms for both ON→OFF logic (conditional inactivation by an RNA trigger) and OFF→ON logic (conditional activation by an RNA trigger). Allosteric cgRNAs enable restriction of CRISPR/Cas function to a desired cell type, tissue, organ, or disease state. Allosteric cgRNAs provide a versatile platform for cell-selective and tissue-selective research tools, biotechnologies, diagnostics, and therapeutics.

Allosteric conditional guide RNAs for cell-selective regulation of CRISPR/Cas

The present application relates to hybridization chain reaction (HCR). In particular, compositions and methods are presented for ultrasensitive molecular detection using HCR signal amplification. Some embodiments and methods involve cooperative probe junctions, reporter-labeled probes, and nonlinear HCR signal amplification.

Ultrasensitive molecular detection via hybridization chain reaction

Programmable guide RNAs (gRNAs) play a central role in the CRISPR revolution sweeping biology and medicine by directing the function of a Cas protein effector to a target gene of choice. To achieve programmable control over regulatory scope, the activity of a conditional guide RNA (cgRNA) depends on the presence or absence of an RNA trigger, allowing for cell-selective regulation of CRISPR/Cas function. Unlike a standard gRNA, a cgRNA is programmable at multiple levels, with the target-binding sequence controlling the target of Cas activity (edit, silence, induce, or bind a gene of choice) and the trigger binding sequence controlling the scope of Cas activity. cgRNA mechanisms that are allosteric allow for independent design of the target and trigger sequences, providing the flexibility to select the regulatory target and scope independently. Disclosed herein are allosteric cgRNA mechanisms for both ON→OFF logic (conditional inactivation by an RNA trigger) and OFF→ON logic (conditional activation by an RNA trigger). Allosteric cgRNAs enable restriction of CRISPR/Cas function to a desired cell type, tissue, organ, or disease state. Allosteric cgRNAs provide a versatile platform for cell-selective and tissue-selective research tools, biotechnologies, diagnostics, and therapeutics.

The present disclosure relates to methods involving HCR reactions that involve using trigger oligos to activate probes that initiate HCR.

Immunohistochemistry via hybridization chain reaction

The present subject matter relates to the use conditional hairpins, such as, but not limited to shRNAs. The conditional formation of these structures can allow for further events, such as gene silencing (in some embodiments).

Small conditional RNAs

The present invention relates generally to programming of biomolecular self-assembly pathways and related methods and constructs for self-assembly of prescribed two and three-dimensional structures.

Biomolecular self-assembly

The present invention relates generally to programming of biomolecular self-assembly pathways and related methods and constructs. A versatile nucleic acid hairpin motif for programming biomolecular self-assembly pathways for a wide variety of dynamic functions, reaction graphs for specifying pathways, and methods of using the hairpin motif are provided.

Versatile nucleic acid hairpin motif for programming biomolecular self-assembly pathways

The present application relates to the use of hybridization chain reaction (HCR) to form double stranded RNA polymers in the presence of a target, such as a nucleic acid associated with a disease or disorder. The RNA polymers are preferably able to activate the RNA-dependent kinase PKR. Activation of PKR via RNA-HCR can be used to treat a wide variety of diseases and disorders by specifically targeting diseased cells.

PKR activation via hybridization chain reaction

The present embodiments relate to engineering imaging probes based on 'triggered molecular geometry.' Upon detection of a molecular signal, nucleic acid hairpin monomers assemble an imageable molecular shape with prescribed geometry. In some embodiments the prescribed shape can be imaged directly. In some embodiments, the prescribed shape can serve as a spatial organizer or amplification scheme for other imaging entities, such as fluorophore and fluorescent proteins.

Triggered molecular geometry based bioimaging probes

The present invention relates to the use of cross-linking probes to covalently bind probes to nucleic acid targets. In some embodiments, the probe comprises an initiator region that is able to bind to a first portion of a target nucleic acid, a probe region linked too the initiator region that is able to bind to a second region of the target nucleic acid and that comprises one or more cross-linkers, and a blocking region hybridized to the probe region.

Triggered covalent probes for imaging and silencing genetic expression

The present invention relates to the use of fluorescently labeled nucleic acid probes to identify and image analytes in a biological sample. In the preferred embodiments, a probe is provided that comprises a target region able to specifically bind an analyte of interest and an initiator region that is able to initiate polymerization of nucleic acid monomers. After contacting a sample with the probe, labeled monomers are provided that form a tethered polymer. Triggered probes and self-quenching monomers can be used to provide active background suppression.

Hybridization chain reaction amplification for in situ imaging

The present application relates to methods and compositions for triggering RNAi. Triggered RNAi is highly versatile because the silencing targets are independent of the detection targets. In some embodiments, methods of silencing or modulating the expression of a target gene are provided. The methods generally comprise providing an initiator to a cell comprising a detection target and a silencing target gene, wherein the detection target is different from the silencing target gene, wherein binding of the detection target to the initiator initiates formation of an inactivator double-stranded RNA (inactivator dsRNA). The inactivator dsRNA can silence or modulate the expression of the silencing target gene.

Growing community of inventors

Pasadena, CA, United States of America

Niles A Pierce