4'-C-[(4-Trifluoromethyl-1H-1,2,3-triazol-1-yl)methyl]thymidine was synthesized and incorporated as a phosphoramidite into oligonucleotide sequences. Its applicability as a sensor for the (19)F NMR spectroscopic detection of DNA and RNA secondary structures was demonstrated. On DNA, the (19)F NMR measurements were focused on monitoring of duplex-triplex conversion, for which this fluorine-labeled 2'-deoxynucleoside proved to be a powerful sensor. This sensor seemingly favors DNA, but its behavior in the RNA environment also turned out to be informative. As a demonstration, invasion of a 2'-O-methyl oligoribonucleotide into an RNA hairpin model (HIV-1 TAR) was monitored by (19)F NMR spectroscopy. According to the thermal denaturation studies by UV spectroscopy, the effect of the 4'-C-(4-trifluoromethyl-1H-1,2,3-triazol-1-yl)methyl moiety on the stability of these DNA and RNA models was marginal.

A sensitive uridine-derived sensor (viz., 2'-O-[(4-CF3-triazol-1-yl)methyl]uridine, 1) for (19)F NMR spectroscopic monitoring of RNA secondary structures is described. The applicability of 1 is demonstrated by monitoring the thermal denaturation of the following double and triple helical RNA models: (1) a miR 215 hairpin, (2) a poly U-A*U triple helix RNA (bearing two C-G*C(H+) interrupts), and (3) a polyadenylated nuclear-nuclear retention element complex. In these RNA models, the (19)F NMR shift of the 2'-O-(CF3-triazolylmethyl) group shows high sensitivity to secondary structural arrangements. Moreover, 1 favors the desired N-conformation, and its effect on both RNA duplex and triplex stabilities is marginal.

2'-O-[(4-Trifluoromethyl-triazol-1-yl)methyl] reporter groups have been incorporated into guanosine-rich RNA models (including a known bistable Qd/Hp RNA and two G-rich regions of mRNA of human prion protein, PrP) and applied for the F NMR spectroscopic characterization of plausible G-quadruplex/hairpin (Qd/Hp) transitions in these RNA structures. For the synthesis of the CF -labeled RNAs, phosphoramidite building blocks of 2'-O-[(4-CF -triazol-1-yl)methyl] nucleosides (cytidine, adenosine, and guanosine) were prepared and used as an integral part of the standard solid-phase RNA synthesis. The obtained F NMR spectra supported the usual characterization data (obtained by UV- and CD-melting profiles and by H NMR spectra of the imino regions) and additionally gave more detailed information on the Qd/Hp transitions. The molar fractions of the secondary structural species (Qd, Hp) upon thermal denaturation and under varying ionic conditions could be determined from the intensities and shifts of the F NMR signals. For a well-behaved Qd/Hp transition, thermodynamic parameters could be extracted.

Triplex-forming peptide nucleic acids (TFPNAs) were targeted to double-helical regions of F-labeled RNA hairpin models (a UA-rich duplex with a hexaethylene glycol (heg) loop and a microRNA model, miR-215). In addition to conventional UV- and circular dichroism (CD)-based detection, binding was monitored by F NMR spectroscopy. Detailed information on the stoichiometry and transition between the triple-helical peptide nucleic acid (PNA)/RNA and (PNA) /RNA binding modes could be obtained. γ-(R)-Hydroxymethyl-modified thymine-1-yl- and 2-aminopyridin-3-yl-acetyl derivatives of TFPNAs were additionally synthesized, which were targeted to the same RNA models, and the effect of the γ-(R)-hydroxymethyl group on binding was studied. An appropriate pattern of γ-(R)-hydroxymethyl modifications reduced the stability of the ternary complex and preferred stoichiometric binding to the miR-215 model.

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