Site-specific bio-conjugation methods and compositions useful for nanopore systems
The present disclosure relates to relates methods and associated compositions that provide fast, efficient site-selective conjugation of a protein, such as the pore-forming protein α-hemolysin, to a biomolecule, such as a DNA polymerase, and the use of such site-selective protein-biomolecule conjugates in nanopore devices and methods.
1. A composition comprising a conjugate of a protein and a biomolecule of formula (Vh) or (Vi):
wherein,
n=1 to 50, and q, r, and s each independently=0, 1, 2, or 3;
L B is a linker;
B is a SpyTaq peptide;
and wherein the protein contains a thiol group
Z is a SpyCatcher protein.
2. The composition of claim 1 , wherein the SpyTag peptide and SpyCatcher protein each comprise a fragment of an amino acid sequence of the CnaB2 domain from the Streptococcus pyogenes fibronectin binding protein FbaB.
3. The composition of claim 1 , wherein the protein is a pore-forming protein, optionally selected from the group consisting of α-hemolysin, β-hemolysin, γ-hemolysin, aerolysin, cytolysin, leukocidin, melittin, MspA porin and porin A.
4. The composition of claim 1 , wherein the protein is a pore-forming protein embedded in a membrane.
5. The composition of claim 1 , wherein the biomolecule is an enzyme capable of catalyzing the synthesis of a polymer, selected from the group consisting of a DNA polymerase, RNA polymerase, reverse transcriptase, and DNA ligase.
6. The composition of claim 1 , wherein the conjugate of formula (Vh) comprises a compound of formula (Vk):
7. The composition of claim 6 , wherein the protein is α-hemolysin and the biomolecule is DNA polymerase.
8. The composition of claim 1 , wherein the conjugate of formula (Vi) comprises a compound of formula (Vm):
9. The composition of claim 8 , wherein the protein is α-hemolysin and the biomolecule is DNA polymerase.