Characterization of G‐Quadruplex/Hairpin Transitions of RNAs by ¹⁹F NMR Spectroscopy

Abstract

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.

The diverse structural landscape of quadruplexes

“…bulged, stem containing long‐loops) could be used as metastable switches , regulating gene expression upon their interaction with different stabilizers inside the cell such as metabolites or other endogenous and exogenous cues . For such a cue‐mediated transition between two kinetically‐accessible structural elements, environmental factors, stem‐loop sequences, cytosine content and combinations thereof are also central players . In addition, intermediates formed during the multistep folding of quadruplexes , for example, trapped stems, may provide structural platforms for gene regulation, driving physiological events.…”

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

“…The ORNs were synthesized on a 1.0 μmol scale by using an Applied Biosystems 3400 DNA/RNA synthesizer, as described previously . Phosphoramidite building blocks of 2′‐ O ‐[(4‐CF 3 ‐triazol‐1‐yl)methyl]uridine and ‐adenosine, together with the commercially available 2′‐ O ‐TBDMS and 2′‐ O ‐Me‐RNA and heg spacer derived building blocks, were used for the chain assembly. Benzylthiotetrazol as an activator and coupling times of 300 (the commercially available building blocks) and 600 s (for 2′‐ O ‐[(4‐CF 3 ‐triazol‐1‐yl)methyl]uridine and ‐adenosine building blocks) were used.…”

“…The ORNs were synthesized on a1 .0 mmol scale by using an Applied Biosystems 3400 DNA/RNA synthesizer,a sd escribed previously. [12] Phosphoramidite building blocks of 2'-O-[(4-CF 3 -triazol-1yl)methyl]uridine [12] and -adenosine, [13] together with the commercially available 2'-O-TBDMS and 2'-O-Me-RNA and heg spacer derived building blocks, were used for the chain assembly.B enzylthiotetrazol as an activator and coupling times of 300 (the commercially available building blocks) and 600 s( for 2'-O-[(4-CF 3 -triazol-1yl)methyl]uridine and -adenosine building blocks) were used. The ORNs were released from the support with am ixture of concentrated ammonia and ethanol [14] (3:1, v/v,3ha t5 58C, overnight at RT),t he silyl protecting groups were removed by treatment with triethylamine trihydrofluoride, [15] and the crude ORNs were purified by RP HPLC to give homogenized ORN1-8 in yields of 8-24 %.…”

“…The spectra were recorded at af requency of 470.6 MHz, as previously described. [12,13] Typical parameters were as follows: 19 Fe xcitation pulse 4.0 ms, acquisition time 1.17 s, prescan delay 6.0 ms, relaxation delay 0.8 s, and the typical number of scans was 2048. The parameters were optimized to gain signals with the longest relaxation rate.…”

Thioflavin T Monitoring of Guanine Quadruplex Formation in the rs689-Dependent INS Intron 1

“…Our results provide insights into the predictive value of G4 scores and highlight the importance of other factors in accurate G4 predictions than a mere presence or absence of G-tracts. Although G-runs are essential for G4 formation,37, 38, 39 other factors are critical, including G-poor loop sequences,40, 41, 42, 43 which may contain accessible antisense targets. The ability of ThT to bind to pockets between adenine pairs 26 would explain the observed high ThT signal for adenine-rich controls, particularly for GA8 and GA12 (Figure 1B).…”