Triplexes with 8‐Aza‐2′‐Deoxyisoguanosine Replacing Protonated dC: Probing Third Strand Stability with a Fluorescent Nucleobase Targeting Duplex DNA

Abstract

The fluorescent 8-aza-2'-deoxyisoguanosine (4) as well as the parent 2'-deoxyisoguanosine (1) were used as protonated dCH(+) surrogates in the third strand of oligonucleotide triplexes. Stable triplexes were formed by Hoogsteen base pairing. In contrast to dC, triplexes containing nucleoside 1 or 4 in place of dCH(+) are already formed under neutral conditions or even at alkaline pH values. Triplex melting can be monitored separately from duplex dissociation in cases in which the third strand contains the fluorescent nucleoside 4. Third-strand binding of oligonucleotides with 4, opposite to dG, was selective as demonstrated by hybridisation experiments studying mismatch discrimination. Third-strand binding is more efficient when the stability of the DNA duplex is reduced by mismatches, giving third-strand binding more flexibility.

Excited-State Proton Transfer and Phototautomerism in Nucleobase and Nucleoside Analogs: A Mini-Review

“…[61,62] By contrast, dual fluorescence has been observed for the product(s) of the slow enzymatic ribosylation of 8-azaisoguanine using purine-nucleoside phosphorylase from E. coli (unpublished observation), most likely indicating ribosylation at N8 (N2 in the IUPAC notation), confirmed independently for this class of compounds. [71] …”

“…[57,58]). In contrast to the natural purines and purine nucleosides, many 8-azapurine congeners exhibit intense room-temperature fluorescence in aqueous medium, [18,59,60] which has been utilized in many biophysical research, including enzyme, [10,11,18] DNA, [61,62] and catalytic RNA studies. [63][64][65] Further inspection into the spectral properties of some 8-azapurines revealed several examples of ESPT and phototautomerism occurring in this class of compounds, briefly summarized previously, [21] and updated below.…”

Fluorescent triplex-forming DNA oligonucleotides labeled with a thiazole orange dimer unit

“…DNA from salmon testes (Sigma-Aldrich) was added to the solution of the D 1 /D 2 duplex (0.4 μM duplex concentration) containing 100 mM sodium phosphate buffer (pH 6) containing 100 mM sodium chloride to be 1, 10 Absorption and fluorescence measurements. Absorption and fluorescence spectra of the ECHO probes (0.4 μM strand concentration) in the absence and presence of their target DNA duplex, D 1 and D 2 (0.4 μM strand concentration), were measured in 100 mM sodium phosphate buffer (pH 6, 7 or 8) containing 100 mM sodium chloride using a cell with a path length of 1 cm.…”

“…Triplex-based fluorescent probes which exhibit changes in fluorescence intensities or wavelengths upon hybridization with a double-stranded DNA have been reported to detect a DNA triplex formation or facilitate the analysis of the thermal stability of DNA triplexes. [6][7][8][9][10][11][12][13][14] Although the probes utilizing Förster resonance energy transfer (FRET) such as molecular beacons could control the emission of the fluorescence, they normally require two kinds of dyes, a fluorophore and quencher, and an extra structure in addition to the sequencerecognition moiety. [6][7][8][9] In contrast, the probes labeling with a single kind of dye have advantages in the facile synthesis and design.…”

“…[6][7][8][9] In contrast, the probes labeling with a single kind of dye have advantages in the facile synthesis and design. [10][11][12][13][14][15] Pyrimidine TFOs labeled with thiazole orange (TO) exhibited comparably larger changes in fluorescence intensities before and after triplex formation 14,15 because the fluorescence intensity of TO molecules is low in the presence of poly-pyrimidine singlestranded DNA 16 and high in the presence of DNA triplexes. 15,17 To suppress the fluorescence of the TO molecules covalently attached to DNA oligonucleotides, the utilization of excitonic interaction between two molecules of TO is effective.…”