Control of Solid-Supported Intra- vs Interstrand Stille Coupling Reactions for Synthesis of DNA-Oligophenylene Conjugates

Abstract

Programmed DNA structures and assemblies are readily accessible, but site-specific functionalization is critical to realize applications in various fields such as nanoelectronics, nanomaterials and biomedicine. Besides pre- and post-DNA synthesis conjugation strategies, on-solid support reactions offer advantages in certain circumstances. We describe on-solid support internucleotide coupling reactions, often considered undesirable, and a workaround strategy to overcome them. Palladium coupling reactions enabled on-solid support intra- and interstrand coupling between single-stranded DNAs (ss-DNAs). Dilution with a capping agent suppressed interstrand coupling, maximizing intrastrand coupling. Alternatively, interstrand coupling actually proved advantageous to provide dimeric organic/DNA conjugates that could be conveniently separated from higher oligomers, and was more favorable with longer terphenyl coupling partners.

Figure 1

On-solid support inter- and intrastrand coupling reactions.

Figure 2

The 20% denaturing polyacrylamide gel electrophoresis (PAGE). (a) Lane M: standard DNA size markers. Lane 1: S₁: 5′-TTT M₁M₁T TTT TTT TTT TTT-3′. Lane 2: coupling product (PP-DNA_n+1) of S₁. Lane 3: coupling product (PP-DNA_n+1) of S₂: 5′-TTT M₁TT M₁TT TTT TTT TTT-3′. Lane 4: S₃: 5′-TM₁T M₁TM₁ TTT TTT TTT TTT-3′. Lane 5: coupling product (PP-DNA_n+1) of S₃. (b) Lane M: standard DNA size markers. Lane 1: S₄: 5′-TTT M₀TT TTT TTT TTT TTT-3′. Lane 2: coupling product (PP-DNA₂ of S₅: 5′-TTT M₂TT TTT TTT TTT TTT-3′. Lane 3: coupling product (PP-DNA₂ of S₆: 5′-TTT M₁TT TTT TTT TTT TTT-3′. (c) Coupling partner C₁. Modified uridines: M₀, M₁ and M₂. Structures of interstrand products PP-DNA_n+1 and PP-DNA₂.

Figure 3

(a) DNA strand: 5′-TTT M₂TT TTT TTT-3′. (b) DNA strand: 5′-TTT M₁TT TTT TTT-3′. Coupling partner C₁.

Figure 4

The 10% nondenaturing PAGE. (a) Lane 1: HB-S₂ monomer. Lane 2: duplex of HB-S₂ monomer with its complement. Lane 3: complementary strand. Lane 4: standard marker. Lane 5: complementary strand. Lane 6: duplex of HB-S₁ monomer with its complement. Lane 7: HB-S₁ monomer. (b) Lane 1: standard marker. Lane 2: complementary strand. Lane 3: duplex of HB-S₂ dimer with its complement. Lane 4: HB-S₂ dimer. ss-DNA strands: HB-S₁: 5′-M₁TG CAG TCT TTT TT-3′; HB-S₂: 5′- M₁TM₁ GCA GTC TTT TTT-3′, Complementary strand: 5′-AAA AAA GAC TGC AAA-3′.

Figure 5

(a) Dilution of ss-DNA on solid support. (b) 20% denaturing PAGE: Lane 1: standard markers. Lane 2 to 5: coupling products of S₇ and C₁. Lane 6 to 9: coupling products of S₈ and C₁. (c) After gel purification, OD₂₆₀ measurement gave relative yields of monomer vs the sum of multimers: with 100% T, the ratio of monomer:dimer:trimer = 58%:30%:12%; with 90% 5′-biotin and 10% T: the ratio of multimers <5%. ss-DNA strands: S₇: 5′-TTT M₁TM₁ TTT XTT TTT TTT-3′; S₈: 5′-TTT M₁TM₁ TTT TTT TTT TTT-3′; X: amidites mixtures (90% 5′-biotin and 10% T).

Figure 6

20% denaturing PAGE: Lane 1: standard markers. Lane 2: starting materials. Lane 3: coupling products with C₂. Lane 4: coupling products with C₃.