1. Protein–Nucleic Acid Conjugation with Sterol Linkers Using Hedgehog Autoprocessing
- Author
-
Timothy S. Owen, Dina S Moumin, José-Luis Giner, Brian P. Callahan, Rebecca A Mancusi, Chunyu Wang, Xiaoyu Zhang, Zihan Xu, and Daniel A Ciulla
- Subjects
Stereochemistry ,Biomedical Engineering ,Pharmaceutical Science ,Bioengineering ,02 engineering and technology ,01 natural sciences ,Article ,chemistry.chemical_compound ,Nucleic Acids ,Animals ,Drosophila Proteins ,Hedgehog Proteins ,Pharmacology ,chemistry.chemical_classification ,DNA ligase ,010405 organic chemistry ,Organic Chemistry ,021001 nanoscience & nanotechnology ,Sterol ,0104 chemical sciences ,Kinetics ,Sterols ,Enzyme ,chemistry ,Glycine ,Nucleic acid ,Drosophila ,0210 nano-technology ,Linker ,DNA ,Biotechnology ,Conjugate - Abstract
Hedgehog (Hh) precursor proteins contain an autoprocessing domain called HhC whose native function is protein cleavage and C-terminal glycine sterylation. The transformation catalyzed by HhC occurs in cis from a precursor protein and exhibits wide tolerance toward both sterol and protein substrates. Here, we repurpose HhC as a 1:1 protein−nucleic acid ligase, with the sterol serving as a molecular linker. A procedure is described for preparing HhC-active sterylated DNA, called steramers, using aqueous compatible chemistry and commercial reagents. Steramers have K(M) values of 7−11 μM and reaction t(1/2) values of ∼10 min. Modularity of the HhC/steramer method is demonstrated using four different proteins along with structured and unstructured sterylated nucleic acids. The resulting protein−DNA conjugates retain the native solution properties and biochemical function. Unlike self-tagging domains, HhC does not remain fused to the conjugate; rather, enzymatic activity is mechanistically coupled to conjugate release. That unique feature of HhC, coupled with efficient kinetics and substrate tolerance, may ease access and open new applications for these suprabiological chimeras.
- Published
- 2019