C2'-pyrene-functionalized triazole-linked DNA: universal DNA/RNA hybridization probes

Abstract

Development of universal hybridization probes, that is, oligonucleotides displaying identical affinity toward matched and mismatched DNA/RNA targets, has been a longstanding goal due to potential applications as degenerate PCR primers and microarray probes. The classic approach toward this end has been the use of "universal bases" that either are based on hydrogen-bonding purine derivatives or aromatic base analogues without hydrogen-bonding capabilities. However, development of probes that result in truly universal hybridization without compromising duplex thermostability has proven challenging. Here we have used the "click reaction" to synthesize four C2'-pyrene-functionalized triazole-linked 2'-deoxyuridine phosphoramidites. We demonstrate that oligodeoxyribonucleotides modified with the corresponding monomers display (a) minimally decreased thermal affinity toward DNA/RNA complements relative to reference strands, (b) highly robust universal hybridization characteristics (average differences in thermal denaturation temperatures of matched vs mismatched duplexes involving monomer W are <1.7 °C), and (c) exceptional affinity toward DNA targets containing abasic sites opposite of the modification site (ΔT(m) up to +25 °C). The latter observation, along with results from absorption and fluorescence spectroscopy, suggests that the pyrene moiety is intercalating into the duplex whereby the opposing nucleotide is pushed into an extrahelical position. These properties render C2'-pyrene-functionalized triazole-linked DNA as promising universal hybridization probes for applications in nucleic acid chemistry and biotechnology.

Figure 1

Structures of C2′-pyrene-functionalized triazole-linked 2′-deoxyuridine monomers and other monomers studied herein.

Figure 2

Representative absorption spectra of single-stranded ON8/ON12/ON16/ON20 (a–d) and their duplexes with matched (M) and centrally mismatched (MM) DNA targets: 3′-GCGTT ABA TTGCG. Nucleotide opposite of modification is mentioned in parenthesis. Spectra were recorded in thermal denaturation buffer at T = 20 °C using 1.0 μM concentration of each strand. Note that different X-axes are used. “O” denotes THF-type abasic site monomer Φ(Figure 1).

Figure 3

Steady-state fluorescence emission spectra of single-stranded ON8/ON12/ON16/ON20 (TTT-context) and duplexes with matched (M) or centrally mismatched (MM) DNA. Recorded in thermal denaturation buffer at T = 20 °C using 1.0 μM of each strand and λ_ex = 350 nm (monomers W, Y and Z) or λ_ex = 400 nm (monomer X). DNA targets 3′-GCGTT ABA TTGCG. Nucleotide opposite of modification is mentioned in parenthesis. Note that different axes are used. “O” denotes THF-type abasic site monomer Φ (Figure 1).

Figure 4

Illustration of putative mechanism resulting in universal hybridization.

Scheme 1

Synthesis and structures of terminal alkynes.

Scheme 2

Synthesis of C2′-pyrene-functionalized triazole-linked uridine phosphoramidites 3V-3Z.