DNA display III. Solid-phase organic synthesis on unprotected DNA.

David R Halpin,Juanghae A Lee,Pehr B Harbury,S Jarrett Wrenn,Gerald Joyce Gerald Joyce

doi:10.1371/journal.pbio.0020175

Abstract

DNA-directed synthesis represents a powerful new tool for molecular discovery. Its ultimate utility, however, hinges upon the diversity of chemical reactions that can be executed in the presence of unprotected DNA. We present a solid-phase reaction format that makes possible the use of standard organic reaction conditions and common reagents to facilitate chemical transformations on unprotected DNA supports. We demonstrate the feasibility of this strategy by comprehensively adapting solid-phase 9-fluorenylmethyoxycarbonyl–based peptide synthesis to be DNA-compatible, and we describe a set of tools for the adaptation of other chemistries. Efficient peptide coupling to DNA was observed for all 33 amino acids tested, and polypeptides as long as 12 amino acids were synthesized on DNA supports. Beyond the direct implications for synthesis of peptide–DNA conjugates, the methods described offer a general strategy for organic synthesis on unprotected DNA. Their employment can facilitate the generation of chemically diverse DNA-encoded molecular populations amenable to in vitro evolution and genetic manipulation.

Highlights

A number of strategies have been proposed recently to enable the in vitro selection and evolution of chemical libraries (Harbury and Halpin 2000; Gartner and Liu 2001). These new approaches to molecular discovery rely upon DNA-directed synthesis, whereby a physical linkage is established between DNA ‘‘genes’’ and respective nonbiological synthetic ‘‘gene products.’’ Such encoded syntheses proceed through a repeated series of two associated steps: reading of sequence information in the DNA and execution of an appropriate chemical transformation
We demonstrate a number of tools for adapting new chemistries, and we use those tools to develop comprehensive methods for peptide synthesis on unprotected DNA
Once each product is covalently attached to an amplifiable support that carries the information necessary for its synthesis, the product molecules are amenable to evolution and genetic manipulation

Summary

Introduction

A number of strategies have been proposed recently to enable the in vitro selection and evolution of chemical libraries (Harbury and Halpin 2000; Gartner and Liu 2001) These new approaches to molecular discovery rely upon DNA-directed synthesis, whereby a physical linkage is established between DNA ‘‘genes’’ and respective nonbiological synthetic ‘‘gene products.’’ Such encoded syntheses proceed through a repeated series of two associated steps: reading of sequence information in the DNA and execution of an appropriate chemical transformation. The strategy has been demonstrated for several types of chemistries (Orgel 1995; Bruick et al 1996; Xu et al 2001; Gartner et al 2002a; Li et al 2002) These proximity methods are necessarily limited to reaction conditions compatible with DNA hybridization, precluding a large number of potential chemical transformations. Only synthetic reagents that have been precoupled to DNA can be used

Methods

Results

Conclusion