Synthesis of Glycosidic (ß-1''→6, 3' and 4') Site Isomers of Neomycin B and their Effect on RNA and DNA Triplex Stability.

Glycosidic (ß-1''→6, 3' and 4') site isomers of neomycin B (i.e., neobiosamine (ß-1''→6, 3' and 4') neamines) have been synthesized in a straightforward manner. Peracetylated neomycin azide was used as a common starting material to obtain neobiosamine glycosyl donor and 6, 3',4'-tri-O-acetyl neamine azide that after simple protecting group manipulation was converted to three different glycosyl acceptors (i.e., 5,6,4'-, 5,3',4'- and 5,6,3'-tri-O-acetyl neamine azide). Glycosylation between the neobiosamine glycosyl donor and the neamine-derived acceptors gave the protected pseudo-tetrasaccharides, which were converted, via global deprotection (deacetylation and reduction of the azide groups), to the desired site isomers of neomycin. The effect of these aminoglycosides on the RNA and DNA triplex stability was studied by UV-melting profile analysis.

Synthesis of Aminoglycoside-2'-O-Methyl Oligoribonucleotide Fusions.

Phosphoramidite building blocks of ribostamycin (3 and 4), that may be incorporated at any position of the oligonucleotide sequence, were synthesized. The building blocks, together with a previously described neomycin-modified solid support, were applied for the preparation of aminoglycoside-2'-O-methyl oligoribonucleotide fusions. The fusions were used to clamp a single strand DNA sequence (a purine-rich strand of c-Myc promoter 1) to form triple helical 2'-O-methyl RNA/DNA-hybrid constructs. The potential of the aminoglycoside moieties to stabilize the triple helical constructs were studied by UV-melting profile analysis.

19 F†NMR Spectroscopic Analysis of the Binding Modes in Triple-Helical Peptide Nucleic Acid (PNA)/MicroRNA Complexes.

Triplex-forming peptide nucleic acids (TFPNAs) were targeted to double-helical regions of 19 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 19 F NMR spectroscopy. Detailed information on the stoichiometry and transition between the triple-helical peptide nucleic acid (PNA)/RNA and (PNA)2 /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.

Characterization of G-Quadruplex/Hairpin Transitions of RNAs by 19 F†NMR Spectroscopy.

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 19 F NMR spectroscopic characterization of plausible G-quadruplex/hairpin (Qd/Hp) transitions in these RNA structures. For the synthesis of the CF3 -labeled RNAs, phosphoramidite building blocks of 2'-O-[(4-CF3 -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 19 F NMR spectra supported the usual characterization data (obtained by UV- and CD-melting profiles and by 1 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 19 F NMR signals. For a well-behaved Qd/Hp transition, thermodynamic parameters could be extracted.

2'-O-[(4-CF3-triazol-1-yl)methyl] Uridine - A Sensitive (19)F NMR Sensor for the Detection of RNA Secondary Structures.

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.

Synthesis of C-5, C-2' and C-4'-neomycin-conjugated triplex forming oligonucleotides and their affinity to DNA-duplexes.

Neomycin-conjugated homopyrimidine oligo 2'-deoxyribonucleotides have been synthesized on a solid phase and their potential as triplex forming oligonucleotides (TFOs) with DNA-duplexes has been studied. For the synthesis of the conjugates, C-5, C-2' and C-4'-tethered alkyne-modified nucleoside derivatives were used as an integral part of the standard automated oligonucleotide chain elongation. An azide-derived neomycin was then conjugated to the incorporated terminal alkynes by Cu(I)-catalyzed 1,3-dipolar cycloaddition (the click chemistry). Concentrated ammonia released the desired conjugates in acceptable purity and yields. The site of conjugation was expectedly important for the Hoogsteen-face recognition: C-5-conjugation showed a notable positive effect, whereas the influence of the C-2' and C-4'-modification remained marginal. In addition to conventional characterization methods (UV- and CD-spectroscopy), (19)F NMR spectroscopy was applied for the monitoring of triplex/duplex/single strand-conversions.

4'-C-[(4-trifluoromethyl-1H-1,2,3-triazol-1-yl)methyl]thymidine as a sensitive (19)F NMR sensor for the detection of oligonucleotide secondary structures.

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.