Zheng-yun Zhao

The folding of the hairpin ribozyme: dependence on the loops and the junction.

In its natural context, the hairpin ribozyme is constructed around a four-way helical junction. This presents the two loops that interact to form the active site on adjacent arms, requiring rotation into an antiparallel structure to bring them into proximity. In the present study we have compared the folding of this form of the ribozyme and subspecies lacking either the loops or the helical junction using fluorescence resonance energy transfer. The complete ribozyme as a four-way junction folds into an antiparallel structure by the cooperative binding of magnesium ions, requiring 20-40 microM for half-maximal extent of folding ([Mg2+]1/2) and a Hill coefficient n = 2. The isolated junction (lacking the loops) also folds into a corresponding antiparallel structure, but does so noncooperatively (n = 1) at a higher magnesium ion concentration ([Mg2+]1/2 = 3 mM). Introduction of a G + 1A mutation into loop A of the ribozyme results in a species with very similar folding to the simple junction, and complete loss of ribozyme activity. Removal of the junction from the ribozyme, replacing it either with a strand break (serving as a hinge) or a GC5 bulge, results in greatly impaired folding, with [Mg2+]1/2 > 20 mM. The results indicate that the natural form of the ribozyme undergoes ion-induced folding by the cooperative formation of an antiparallel junction and loop-loop interaction to generate the active form of the ribozyme. The four-way junction thus provides a scaffold in the natural RNA that facilitates the folding of the ribozyme into the active form.

Mass Determination of Phosphoramidites

Nucleoside phosphoramidites are the most widely used building blocks in contemporary solid-phase synthesis of oligonucleotides. The accurate molecular weight measurements of such molecules, which are acid-labile compounds, may be easily determined by mass spectrometry using a matrix system, triethanolamine/NaCl, on a liquid secondary ion mass spectrometer (LSIMS) or fast-atom bombardment (FAB) MS equipped with a double-focusing mass spectrometer. The present method rapidly and easily measures the accurate molecular weights of various phosphoramidites as adduct ions [M+Na]+ with an average mass error smaller than 0.4 ppm, allowing determination of the formulas of the phosphoramidites in place of elemental analysis. Further, it was found that intensities of molecular-related ions could be enhanced to the highest degree by adjustment of the molar ratio of phosphoramidite and NaCl, fixing the amount of triethanolamine on LSIMS, making the present method a powerful tool for identification of phosphoramidites by mass spectrometry.

Synthesis of Triazol Cn-Ribonucleoside Phosphoramidites Using β-Ribofuranosyl-Cn-acetylenes for RNA Catalysis Probing

Novel C4-linked triazol C0-, C1and C2-ribonucleoside phosphoramidites for RNA catalysis probing were synthesized from β-ribofuranosyl-Cn-acetylenes (n = 0–2), which were efficiently prepared by fragmentation of tetrazoles derived from cyanophosphates. N-Pivaloyloxymethyl moiety was selected for the protection of triazole-N, whose properties under acidic and basic conditions were investigated. INTRODUCTION We have recently reported the efficient synthesis of C4-linked C0to C3-imidazole ribonucleoside phosphoramidites [Imz-Cn-PAs (1a–d)], which could be introduced in RNA using solid-phase t-BDMS chemistry implemented on an automated synthesizer, as shown in Figure 1.1 During the synthesis of these phosphoramidites, pivaloyloxymethyl (POM)1b and cyanoethyl (CE)1c groups were employed successfully as suitable protecting groups for the imidazole -nitrogen and sugar 2’-hydroxy functions, respectively. Since imidazoles (pKa of 14.2) are both good proton donors and acceptors,2 we developed a novel chemogenetic approach using Imz-Cn-PAs 1 for the study of the catalytic mechanism of Varkud satellite (VS)3a and hairpin ribozymes,3b where conventional nucleobases are replaced by imidazoles as a powerful tool to probe general acid–base catalysis in the active sites of ribozymes.4 The results from this approach indicated that the chemical mechanisms of VS and hairpin ribozymes involve general acid–base catalysis via a combination of specific adenine (A) and guanine (G) nucleobases (e.g., A756 and G638 in 106 HETEROCYCLES, Vol. 96, No. 1, 2018