Figure 1.
Peptide Coupling to DNA Supports
(A) Fmoc-based peptide coupling reaction to an aminated 20-base oligonucleotide (NC20) where X represents a succinimidyl or EDC/HOAt-activated ester.
(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).
(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).
Figure 2.
DNA Support Structure and Modified Amino Acids
(A) Peptide synthesis is carried out on DNA modified with a 5′ C12 amino (NC20) or a 5′ PEG amino (NP20) linker.
(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).
Table 1.
Amino Acid Coupling Efficiencies to Leu-NC20
Figure 3.
Peptide–DNA Conjugate As Template for DNA Synthesis
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.
Table 2.
Sequenced Peptide–DNA Conjugates