Reactivity-dependent PCR: direct, solution-phase in vitro selection for bond formation.
In vitro selection is a key component of efforts to discover functional nucleic acids and small molecules from libraries of DNA, RNA, and DNA-encoded small molecules. Such selections have been widely used to evolve RNA and DNA catalysts and, more recently, to discover new reactions from DNA-encoded libraries of potential substrates. While effective, current strategies for selections of bond-forming and bond-cleaving reactivity are generally indirect, require the synthesis of biotin-linked substrates, and involve multiple solution-phase and solid-phase manipulations. In this work we report the successful development and validation of reactivity-dependent PCR (RDPCR), a new method that more directly links bond formation or bond cleavage with the amplification of desired sequences and that obviates the need for solid-phase capture, washing, and elution steps. We show that RDPCR can be used to select for bond formation in the context of reaction discovery and for bond cleavage in the context of protease activity profiling.
Gorin
David J
DJ
Howard Hughes Medical Institute and Department of Chemistry and Chemical Biology, Harvard University, Cambridge, Massachusetts 02138, USA.
Kamlet
Adam S
AS
Liu
David R
DR
D004247:DNA / Q000737:chemistry* / Q000235:genetics; D019020:Directed Molecular Evolution / Q000191:economics / Q000379:methods*; D015723:Gene Library; D055029:Inverted Repeat Sequences; D016133:Polymerase Chain Reaction / Q000191:economics / Q000379:methods*; D012313:RNA / Q000737:chemistry* / Q000235:genetics; D044366:Transition Temperature
D012313:RNA; D004247:DNA