C2′-Pyrene-Functionalized Triazole-Linked DNA: Universal DNA/RNA Hybridization Probes

Abstract

Development of universal hybridization probes, i.e., 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 aromatic base analogs without hydrogen-bonding capabilities or hydrogen-bonding purine derivatives. However, development of probes that enable 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 are < 1.5 °C); and c) exceptional affinity toward DNA targets containing abasic sites opposite of the modification site (ΔTm up to +25 °C). The latter observation, along with results from absorption and fluorescence spectroscopy, indicates 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.

Aptamers Chemistry: Chemical Modifications and Conjugation Strategies

“…Triazole-modified DNA aptamers with a structure similar to thrombin-inhibiting G-quadruplexes, TBA15 and TBA31, had been tested for their stabilities and binding affinities. No change was observed in their binding affinities, but the triazole modification protected aptamers from nuclease hydrolysis and increased their stabilities [110,115].…”

“…Oligonucleotide triazole modification instead of phosphodiester linkage has been investigated extensively in many studies [109,110] to protect the oligonucleotides from nuclease hydrolysis [111].…”

Metal‐Free Route for the Synthesis of 4‐Acyl‐1,2,3‐Triazoles from Readily Available Building Blocks

“…It is well documented that α‐ketotriazole building blocks display various interesting biological properties (Figure ) . Recently, these compounds have been employed as hybridization probes for applications in nucleic acid chemistry, such as the development of triazole‐linked DNA …”

“…The application of this strategy to the α,β‐unsaturated ketone 13 led to the synthesis of a chalcone derivative 14 with excellent yield (Scheme ). These classes of compounds were shown to have reversible tissue transglutaminase inhibitor activity and are usually synthesized using a multistep strategy . Interestingly, the reaction also worked well with 3‐acetylcoumarin 15 , which afforded the useful material 16 .…”

Fluorescent Nucleic Acid Analogues in Research and Clinical Diagnostics

“…In doing this, the choice of a fluorophore becomes a critical factor. The optimal groove-binding fluorophore is highly sensitive to the local microenvironment, stable under conditions of oligonucleotide synthesis or postsynthetic labeling, water soluble, and bright (Lindegaard et al 2008;Umemoto et al 2007;Astakhova et al 2008aAstakhova et al , b, 2010Astakhova et al , 2012Astakhova et al , 2013aAstakhova 2014;Østergaard et al 2010a, b;Sau and Hrdlicka 2012;Kumar et al 2013). Notably, the chemistry and length of a linker play an important role for the attachment of groove-binding dyes to oligonucleotides.…”