DNA Display III. Solid-Phase Organic Synthesis on Unprotected DNA

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

Peptide–DNA Conjugate As Template for DNA Synthesis

<p>5′ PEG amino-modified 340-base ssDNA was loaded onto two DEAE Sepharose columns. The pentapeptide [Leu]enkephalin was synthesized on one column using EDC/HOAt and Fmoc–amino acids. The DNA was eluted, desalted, and used as template for radiolabeled primer extension reactions. Denaturing polyacrylamide gel electrophoresis analysis of reaction products shows no difference between ssDNA (control) and [Leu]enkephalin–ssDNA ([Leu]enk) templates.</p

DNA Support Structure and Modified Amino Acids

<div><p>(A) Peptide synthesis is carried out on DNA modified with a 5′ C12 amino (NC20) or a 5′ PEG amino (NP20) linker.</p> <p>(B) Fluorescent lysine derivative (compound 1, Fmoc-Lys[coumarin]-OH) and BME/DBU labile protecting groups for lysine (compound 2, Fmoc-Lys[Ns]-OH), arginine (compound 3, Fmoc-Arg[Ns]-OH), and histidine (compound 4, Fmoc-His[CNP]-OH).</p></div

Peptide Coupling to DNA Supports

<div><p>(A) Fmoc-based peptide coupling reaction to an aminated 20-base oligonucleotide (NC20) where X represents a succinimidyl or EDC/HOAt-activated ester.</p> <p>(B) HPLC chromatograms of a nonaminated 10-base (10mer) and an aminated 20-base (NC20) oligonucleotide. HPLC traces show DEAE column load (solid black) and elute (broken black). DEAE column elutes after succinimidyl ester–mediated (solid red) or EDC/HOAt-mediated (broken red) Fmoc-Leu coupling and Fmoc deprotection are shown. The resulting amino acid–DNA conjugate is denoted (Leu-NC20).</p> <p>(C) Chemical transformations are carried out using small DEAE Sepharose columns and syringes (left). Washes are facilitated by a vacuum manifold with chemically resistant stopcocks (right).</p></div