Compositions, systems and methods for CRISPR-based enzyme optimization
Provided herein are reaction mixtures, compositions, systems, methods, and kits for assessing enzymatic activity. Aspects of the disclosure include use of a reactant barcoded oligonucleotide (RBO) construct comprising a first oligonucleotide comprising a reaction barcode sequence and a first linker that connects the first oligonucleotide to a first reactant, an amplification 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 a second reactant, and a blocking construct, comprising a third oligonucleotide that is complementary to at least a portion of the first oligonucleotide. A rate of reaction product generation may be measured to determine an enzymatic activity of a given enzyme.
1 . A method for assessing enzymatic activity, the method comprising: combining a reactant barcoded oligonucleotide (RBO) construct with an amplification construct, a blocking construct, and an enzyme in a reaction mixture, wherein the reactant barcoded oligonucleotide (RBO) construct comprises:
a first oligonucleotide comprising a reaction barcode sequence; and a first linker that connects the first oligonucleotide to a first reactant;
wherein the amplification construct comprises:
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 reactant; and
wherein the blocking construct comprises:
a third oligonucleotide that is complementary to at least a portion of the first oligonucleotide;
enzymatically reacting the first reactant with the second reactant to produce a reaction product;
restricting movement of the amplification construct relative to the RBO construct; and
generating a reverse complement of the reaction barcode sequence with an amplification enzyme.
2 . The method of claim 1 , wherein generating a reverse complement of the reaction barcode sequence with an amplification enzyme comprises conducting an amplification reaction on the first oligonucleotide comprising the reaction barcode sequence to produce an extension product.
3 . The method of claim 2 , wherein the amplification reaction comprises the step of annealing a second adapter to a second adapter binding site adjacent to a 3′ end of the reaction barcode sequence and generating a reverse complement of the reaction barcode sequence.
4 . The method of claim 3 , wherein the step of annealing further comprises annealing a first adapter to a first adapter binding site adjacent to a 5′ end of the barcode sequence.
5 . The method according to claim 2 , wherein the amplification enzyme comprises a polymerase.
6 . The method according to claim 2 , wherein the amplification enzyme comprises a reverse transcriptase.
7 . The method according to claim 1 , further comprising:
assessing an enzymatic activity of an enzyme based on an amplification product of the amplification reaction.
8 . The method of claim 7 , wherein the amplification product is detectable.
9 . The method of claim 7 , wherein the amplification product comprises a detectable label.
10 . The method of claim 7 , wherein the amplification product encodes a detectable label.
11 . The method according to claim 2 , further comprising:
identifying the enzyme using the reaction barcode sequence.
12 . The method according to claim 2 , further comprising:
modifying an amino acid sequence of the enzyme.
13 . The method of claim 12 , wherein the step of modifying occurs before combining the reactant barcoded oligonucleotide (RBO) construct with the amplification construct, the blocking construct, and the enzyme in the reaction mixture.
14 . The method according claim 12 , wherein modifying an amino acid sequence of the enzyme comprises a CRISPR-mediated amino acid sequence modification.
15 . The method according to claim 1 , wherein the first and second reactants each comprise a protein.
16 . The method according to claim 1 , wherein the first and second reactants each comprise a polynucleotide.
17 . The method according to claim 1 , further comprising:
increasing an affinity between the RBO construct and the blocking construct.
18 . The method according to claim 1 , wherein the second oligonucleotide comprises at least one LNA.
19 . The method according to claim 1 , wherein the portion of the first oligonucleotide that is complementary to the second oligonucleotide comprises at least one LNA.
20 . The method according to claim 1 , wherein the second oligonucleotide comprises a non-reactive 3′ end.
21 . The method according to claim 1 , wherein the reaction product comprises the first reactant covalently linked to the second reactant.
22 . The method according to claim 1 , wherein a quantity of the blocking construct is greater than a quantity of the amplification construct.