Method and Composition for Biocatalytic Protein-Oligonucleotide Conjugation

Background:

Site-specific selective protein modification procedures have been useful for oriented protein immobilization, studies of naturally occurring post-translational modifications, creating antibody-drug conjugates, introduction of fluorophores and other small molecules on to proteins, examining protein structure, dynamics, interactions, and for the preparation of protein-polymer conjugates. Of specific interest is the conjugation of proteins to nucleic acids. Current methods to conjugate proteins with nucleic acids depend on “spontaneous” chemical conjugation chemistry, such as disulfide bond formation, as opposed to conjugation catalyzed by a biomolecule. The methods in current use require installing reactive functional groups on the protein and separately, on the nucleic acid. 

 Technology Overview:

The present technology provides a novel means to catalytically ligate a polypeptide to a nucleic acid at a defined site with 1:1 stoichiometry in a single step. This new method is meant to replace traditional protein-nucleic acid conjugation via spontaneous coupling chemistries, which are often inefficient and nonselective. The catalyst is a modular autoprocessing protein called hedgehog sterol transferase (HST-1). Expressed as a transient fusion to the protein of interest, HST-1 catalyzes ligation through co-factor independent transacylation. Typical ligations take place in under 1 hour at neutral pH, 25 degrees Celsius. Importantly, HST-1 is released from the protein of interest upon ligation, rendering the reaction traceless. Because of its unique mechanism of action, HST-I may find multiple applications in the field of bioconjugation, particularly in the areas of therapeutics and diagnostics. Site-specific selective protein modification by HST-1 may also prove useful for oriented protein immobilization, creating antibody-drug conjugates, introduction of fluorophores and other small molecules onto proteins, examining protein interactions and for the preparation of protein-polymer conjugates.

 Advantages:

• ligation to nucleic acids (DNA, RNA), various small molecules
• 1:1 stoichiometry
• Single-step, cofactor-independent reaction
• Under 1 hour at neutral pH and 25℃.
• Catalyzed by hedgehog sterol transferase (HST-1), released upon ligation
• Conjugates retain native solution properties and biochemical function

 

Intellectual Property Summary:

• U.S. 10,738,338