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US 2024/0191291 A1Published application
Compositions, systems and methods for enzyme optimization
Assignee10x Genomics, Inc.
InventorMichael GIBBONS
Life of this patent
Estimating the term…
Abstract
Provided herein are reaction mixtures, compositions, systems, methods, and kits for assessing enzymatic activity. Aspects of the disclosure include use of a cleavable substrate comprising a first portion linked to a second portion designed to be the target of an enzyme. A substrate barcoded oligonucleotide (SBO) construct may be linked to the first portion and a blocking construct may be linked to the second portion. A rate of substrate cleavage may be measured to determine an enzymatic activity of a given enzyme.
Claims100
A method of assessing enzymatic activity within a partition, the method comprising:
- (i) providing a partition comprising a substrate complex and an enzyme, wherein the substrate complex comprises:
- a cleavable substrate comprising a first portion linked to a second portion;
- a substrate barcoded oligonucleotide (SBO) construct, comprising:
- a first oligonucleotide comprising a barcode sequence that identifies the cleavable substrate;
- and
- a first linker that connects the first oligonucleotide to the first portion of the substrate;
- a blocking construct, comprising a second oligonucleotide that is complementary to at least a portion of the first oligonucleotide;
- and
- a second linker that connects the second oligonucleotide to the second portion of the cleavable substrate;
- (ii) restricting movement of the blocking construct relative to the SBO construct to inhibit generation of a reverse complement of the barcode sequence with the blocking constructs;
- (iii) cleaving the cleavable substrate with the enzyme;
- (iv) releasing the blocking construct from the SBO construct;
- (v) conducting an amplification reaction on the first oligonucleotide comprising the barcode sequence;
- and
- (vi) assessing enzymatic activity based on a reaction product of the amplification reaction.
12 more elements
A reaction mixture for assessing enzymatic activity, the reaction mixture comprising:
- (i) a cleavable substrate comprising a first portion linked to a second portion;
- (ii) a substrate barcoded oligonucleotide (SBO) construct, comprising:
- a first oligonucleotide comprising a barcode sequence;
- and
- a first linker that connects the first oligonucleotide to the first portion of the substrate;
- (iii) a blocking construct, comprising:
- a second oligonucleotide that is complementary to at least a portion of the first oligonucleotide;
- and
- a second linker that connects the second oligonucleotide to the second portion of the substrate.
6 more elements
A reaction mixture for assessing enzymatic activity, the reaction mixture comprising:
- a substrate complex, comprising:
- (i) a plurality of cleavable substrates, each comprising a first portion linked to a second portion;
- (ii) a plurality of SBO constructs, each comprising:
- a first oligonucleotide comprising a substrate barcode sequence that identifies the substrate;
- and
- a first linker that connects the first oligonucleotide to a first portion of one of the substrates;
- and
- (iii) a plurality of blocking constructs, each comprising:
- a second oligonucleotide that is complementary to at least a portion of the first oligonucleotide;
- and
- a second linker that connects the second oligonucleotide to a second portion of one of the substrates.
8 more elements
A method for assessing enzymatic activity, the method comprising:
- (i) contacting a substrate complex with an enzyme, wherein the substrate complex comprises:
- a cleavable substrate comprising a first portion linked to a second portion;
- a substrate barcoded oligonucleotide (SBO) construct, comprising:
- a first oligonucleotide comprising a barcode sequence;
- and
- a first linker that connects the first oligonucleotide to the first portion of the substrate;
- a blocking construct, comprising a second oligonucleotide that is complementary to at least a portion of the first oligonucleotide;
- and
- a second linker that connects the second oligonucleotide to the second portion of the substrate;
- (ii) restricting movement of the blocking construct relative to the SBO construct to inhibit generation of a reverse complement of the barcode sequence with the blocking construct;
- (iii) cleaving the cleavable substrate with the enzyme;
- and
- (iv) releasing the blocking construct from the SBO construct.
10 more elements
A method for assessing and comparing enzymatic activity, the method comprising:
- (i) contacting a plurality of unique substrate complexes with a plurality of corresponding unique enzymes, wherein each of the plurality of unique substrate complexes comprises:
- a unique cleavable substrate comprising a first portion linked to a second portion;
- a unique substrate barcoded oligonucleotide (SBO) construct, comprising:
- a first oligonucleotide comprising a substrate barcode sequence that identifies the unique cleavable substrate;
- and
- a first linker that connects the first oligonucleotide to the first portion of the substrate;
- a blocking construct, comprising:
- a second oligonucleotide that is complementary to at least a portion of the first oligonucleotide;
- and
- a second linker that connects the second oligonucleotide to the second portion of the substrate;
- (ii) restricting movement of the blocking constructs relative to the unique SBO constructs to inhibit generation of a reverse complement of the substrate barcode sequences with the blocking constructs;
- (iii) cleaving one or more of the unique substrates with its corresponding unique enzyme;
- (iv) releasing one or more of the blocking constructs from the unique SBO constructs;
- (v) conducting an amplification reaction on the first oligonucleotides comprising the substrate barcode sequences;
- and
- (vi) assessing enzymatic activity based on a reaction product of the amplification reaction.
13 more elements
A partition, comprising:
- (i) a cleavable substrate comprising a first portion linked to a second portion;
- (ii) a substrate barcoded oligonucleotide (SBO) construct, comprising:
- a first oligonucleotide comprising a barcode sequence that identifies the cleavable substrate;
- and
- a first linker that connects the first oligonucleotide to the first portion of the substrate;
- (iii) a blocking construct, comprising:
- a second oligonucleotide that is complementary to at least a portion of the first oligonucleotide;
- and
- a second linker that connects the second oligonucleotide to the second portion of the substrate.
6 more elements
A kit comprising:
- (i) a cleavable substrate comprising a first portion linked to a second portion;
- (ii) a substrate barcoded oligonucleotide (SBO) construct, comprising:
- a first oligonucleotide comprising a barcode sequence that identifies the cleavable substrate;
- and
- a first linker that connects the first oligonucleotide to the first portion of the substrate;
- (iii) a blocking construct, comprising:
- a second oligonucleotide that is complementary to at least a portion of the first oligonucleotide;
- and
- a second linker that connects the second oligonucleotide to the second portion of the substrate.
6 more elements
Dependencies are inferred from the claim text.