Catalytic biomolecule activity recording into DNA sequence
The present invention relates to catalytic biomolecule characterization and microfluidics. It is used for identification of nucleic acids encoding active catalytic molecules in the plurality of nucleic acids and for gathering information about catalytic biomolecule activity. It can also be used for exploring different properties of regulating sequences that modulate expression of catalytic biomolecules by recording that information into the DNA sequence of the same catalytic biomolecule using microfluidic techniques.
1. A method for identifying a nucleic acid encoding an active catalytic molecule in a plurality of nucleic acids, comprising:
providing a plurality of compartments, each compartment comprising no more than one nucleic acid molecule encoding a catalytic biomolecule,
subjecting the compartments to conditions suitable for synthesis of the catalytic biomolecule,
providing substrate nucleotide to the compartments for said catalytic biomolecule,
wherein the substrate nucleotide cannot be incorporated into synthesized nucleic acid by polymerase,
subjecting said compartments to reaction conditions that allow catalytic molecule enzymatic activity to occur,
wherein said activity results in the modification of the substrate nucleotide, so that the nucleotide becomes suitable for incorporating into synthesized nucleic acid by polymerase,
providing into said compartments nucleic acid synthesis reaction mixture comprising reference nucleotides producing at least one copy of nucleic acid contained in the compartment,
wherein modified substrate nucleotide and reference nucleotide are incorporated into nucleic acid sequence by polymerase concurrently,
determining the ratio between reference nucleotide and modified substrate nucleotide in a newly synthesized nucleic acid,
wherein said ratio is indicative of catalytic activity of the catalytic biomolecule encoded by said nucleic acid.
2. The method according to claim 1 , wherein the plurality of nucleic acids is obtained by a method selected from error-prone PCR, oligonucleotide-directed mutagenesis, assembly PCR, in vivo mutagenesis, cassette mutagenesis, recursive ensemble mutagenesis, exponential ensemble mutagenesis and site-specific mutagenesis or by lysis of a plurality of cells.
3. The method according to claim 1 , wherein the compartment is a droplet, a microwell, a microtube, or an individual spot on a slide.
4. The method according to claim 1 , wherein the reference nucleotide is 5-methyl-2′-deoxycytidine triphosphate.
5. The method according to claim 1 , wherein substrate nucleotide is N4-benzoyl-2′-deoxycytidine triphosphate.
6. The method according to claim 1 , wherein catalytic biomolecule is an enzyme, ribozyme or deoxyribozyme.
7. The method according to claim 6 , wherein catalytic biomolecule is an esterase enzyme.
8. The method according to claim 1 , wherein catalytic biomolecule catalytic activity is oxidoreduction, transfer of a chemical group, hydrolysis, bond cleavage other than hydrolysis or oxidation, changes within a single molecule, or joining two molecules with concomitant hydrolysis of the diphosphate bond in ATP.
9. The method according to claim 8 , wherein catalytic biomolecule activity is cleavage of ester bond.
10. The method according to claim 1 , wherein producing at least one copy of nucleic acid is performed by strand elongation, isothermal amplification reaction, or polymerase chain reaction.
11. The method according to claim 1 , wherein ratio between reference nucleotide and modified substrate nucleotide in a newly synthesized nucleic acid is determined by single nucleic acid molecule analysis methods.
12. The method according to claim 11 , wherein single nucleic acid molecule analysis method includes Nanopore sequencing, Bisulfite sequencing, Tethered Oligonucleotide-Primed sequencing, or Single Molecule Real Time (SMRT) sequencing.
13. The method according to claim 12 , wherein single nucleic acid molecule analysis method is able to detect different nucleotide modifications.
14. The method according to claim 1 , wherein synthesis of catalytic biomolecule is regulated by genetic regulatory sequences.
15. The method according to claim 14 , wherein genetic regulatory sequence is ribosome binding site, promoter sequence, ribozyme, riboswitch, transcription enhancer, operator, repressor binding site, activator binding site, DNA binding site, terminator, repressor encoding gene, or activator encoding gene.
16. The method of claim 1 , wherein providing substrate nucleotide to the compartments is done before or after the step of subjecting the compartments to conditions suitable for synthesis of the catalytic biomolecule.
17. A method for identifying a nucleic acid encoding an active catalytic molecule in a plurality of nucleic acids, comprising:
providing a plurality of compartments, each compartment comprising no more than one nucleic acid molecule encoding a catalytic biomolecule,
subjecting the compartments to conditions suitable for synthesis of the catalytic biomolecule,
providing substrate nucleotide to the compartments for said catalytic biomolecule,
wherein the substrate nucleotide can be incorporated into synthesized nucleic acid by polymerase,
subjecting said compartments to reaction conditions that allow catalytic molecule enzymatic activity to occur,
wherein said activity results in the modification of the substrate nucleotide, so that the nucleotide becomes no longer suitable for incorporating into synthesized nucleic acid by polymerase,
providing into said compartments nucleic acid synthesis reaction mixture comprising reference nucleotides
producing at least one copy of nucleic acid contained in the compartment,
wherein substrate nucleotide and reference nucleotide are incorporated into nucleic acid sequence by polymerase concurrently,
determining the ratio between reference nucleotide and substrate nucleotide in a newly synthesized nucleic acid,
wherein said ratio is indicative of catalytic activity of the catalytic biomolecule encoded by said nucleic acid.
18. The method of claim 17 , wherein providing substrate nucleotide to the compartments is done before or after the step of subjecting the compartments to conditions suitable for synthesis of the catalytic biomolecule.
19. A method for identifying a nucleic acid encoding an active catalytic molecule in a plurality of nucleic acids, comprising:
providing a plurality of compartments, each compartment comprising no more than one nucleic acid molecule encoding a catalytic biomolecule,
subjecting the compartments to conditions suitable for synthesis of the catalytic biomolecule,
providing substrate nucleotide to the compartments for said catalytic biomolecule,
subjecting said compartments to reaction conditions that allow catalytic molecule enzymatic activity to occur,
wherein said activity results in the modification of the substrate nucleotide,
providing into said compartments nucleic acid synthesis reaction mixture comprising reference nucleotides
producing at least one copy of nucleic acid contained in the compartment,
wherein substrate nucleotide, modified substrate nucleotide and reference nucleotide are incorporated into nucleic acid sequence by polymerase concurrently,
determining the ratio between reference nucleotide, substrate nucleotide and modified substrate nucleotide in a newly synthesized nucleic acid,
wherein said ratio is indicative of catalytic activity of the produced catalytic biomolecule.
20. The method of claim 19 , wherein providing substrate nucleotide to the compartments is done before or after the step of subjecting the compartments to conditions suitable for synthesis of the catalytic biomolecule.