Pyrene‐Modified Unlocked Nucleic Acids: Synthesis, Thermodynamic Studies, and Fluorescent Properties

Abstract

Two pyrene-modified UNA monomers were synthesized and incorporated into 21-mer DNA oligonucleotides. Melting temperatures and thermodynamic properties of the modified duplexes were measured, and the fluorescence properties of single strands and duplexes containing one or more pyrene-UNA modifications were studied. It was found that incorporation of pyrene-UNA monomers increased duplex stability relative to UNA monomers, and thermodynamic studies revealed significant mismatch discriminative capabilities of the pyrene-UNA modified oligonucleotides. Furthermore, the steady-state fluorescence emission intensities of pyrene-UNA modified oligonucleotides were increased upon hybridization to DNA, which to the best of our knowledge is unprecedented for an acyclic pyrene modification in DNA. Interestingly, pyrene excimer emission was observed for single-stranded oligonucleotides containing three pyrene-UNA modifications, whereas this excimer emission disappeared after hybridization to DNA. In view of both the pyrene monomer and the excimer fluorescence emission, the triply modified oligonucleotides show intriguing properties relating to the development of new DNA/RNA detection tools.

Unlocked Nucleic Acids with a Pyrene‐Modified Uracil: Synthesis, Hybridization Studies, Fluorescent Properties and i‐Motif Stability

“…Opening of the furanose ring leads to increased flexibility in an UNA monomer. This has a negative effect on the stabilities of nucleic acid duplexes,2–6 but can improve mismatch discrimination 3. 6 UNA monomers were also found to modulate the stabilities of i‐motif7 and G‐quadruplex8 structures.…”

“…This has a negative effect on the stabilities of nucleic acid duplexes,2–6 but can improve mismatch discrimination 3. 6 UNA monomers were also found to modulate the stabilities of i‐motif7 and G‐quadruplex8 structures. UNA monomers therefore meet the requirements for applications in antisense oligonucleotides,9, 10 siRNA constructs11 and aptamer development 12.…”

“…Chemical synthesis of UNA phosphoramidite building blocks is simple,2, 3 and an UNA monomer can be incorporated into oligonucleotides either through the O3′2, 3 or the O2′2, 4, 9 atom. The sugar moiety of an UNA monomer can easily be conjugated with, for example, ligands for complexation of metal ions13 or attachment of fluorescent labels6 such as pyrene.…”

Fluorescent Nucleic Acid Analogues in Research and Clinical Diagnostics

“…Typically, dye intercalation results in increased thermal stability of the double helix and altered fluorescence signal of the dye (Glazer and Rye 1992;Berndl and Wagenknecht 2009). Intercalators can be either attached covalently to the nucleic acid sequence or interact with the duplex non-covalently (Rye and Glazer 1995;Karlsen et al 2012). Non-nucleosidic analogues of nucleic acids such as IPN, UNA, and TINA contain a covalently attached intercalating dye within the oligonucleotide probe which is labeled during oligonucleotide synthesis or postsynthetically (Filichev and Pedersen 2005;El-Sayed et al 2012;Stadler et al 2011).…”

Non-nucleosidic analogs of nucleic acids

“…The derivatives with a piperazine group (Karlsen et al, 2009;Karlsen et al, 2012) are among the possible linkers of useful ligands, including a terpyridine ligand for metal chelation (Karlsen et al, 2009;Karlsen et al, 2012).…”