Understanding In-line Probing Experiments by Modeling Cleavage of Non-reactive RNA Nucleotides

Ribonucleic acid (RNA) is involved in many regulatory and catalytic processes in the cell. The function of any RNA molecule is intimately related with its structure. In-line probing experiments provide valuable structural datasets for a variety of RNAs and are used to characterize conformational changes in riboswitches. However, the structural determinants that lead to differential reactivities in unpaired nucleotides have not been investigated yet. In this work we used a combination of theoretical approaches, i.e., classical molecular dynamics simulations, multiscale quantum mechanical/molecular mechanical calculations, and enhanced sampling techniques in order to compute and interpret the differential reactivity of individual residues in several RNA motifs including members of the most important GNRA and UNCG tetraloop families. Simulations on the multi ns timescale are required to converge the related free-energy landscapes. The results for uGAAAg and cUUCGg tetraloops and double helices are compared with available data from in-line probing experiments and show that the introduced technique is able to distinguish between nucleotides of the uGAAAg tetraloop based on their structural predispositions towards phosphodiester backbone cleavage. For the cUUCGg tetraloop, more advanced ab initio calculations would be required. This study is the first attempt to computationally classify chemical probing experiments and paves the way for an identification of tertiary structures based on the measured reactivity of non-reactive nucleotides

Chemical feasibility of the general acid/base mechanism of glmS ribozyme self‐cleavage

In numerous Gram‐positive bacteria, the glmS ribozyme or catalytic riboswitch regulates the expression of glucosamine‐6‐phosphate (GlcN6P) synthase via site‐specific cleavage of its sugar‐phosphate backbone in response to GlcN6P ligand binding. Biochemical data have suggested a crucial catalytic role for an active site guanine (G40 in Thermoanaerobacter tengcongensis, G33 in Bacillus anthracis). We used hybrid quantum chemical/molecular mechanical (QM/MM) calculations to probe the mechanism where G40 is deprotonated and acts as a general base. The calculations suggest that the deprotonated guanine G40− is sufficiently reactive to overcome the thermodynamic penalty arising from its rare protonation state, and thus is able to activate the A‐1(2′‐OH) group toward nucleophilic attack on the adjacent backbone. Furthermore, deprotonation of A‐1(2′‐OH) and nucleophilic attack are predicted to occur as separate steps, where activation of A‐1(2′‐OH) precedes nucleophilic attack. Conversely, the transition state associated with the rate‐determining step corresponds to concurrent nucleophilic attack and protonation of the G1(O5′) leaving group by the ammonium moiety of the GlcN6P cofactor. Overall, our calculations help to explain the crucial roles of G40 (as a general base) and GlcN6P (as a general acid) during glmS ribozyme self‐cleavage. In addition, we show that the QM/MM description of the glmS ribozyme self‐cleavage reaction is significantly more sensitive to the size of the QM region and the quality of the QM‐MM coupling than that of other small ribozymes. © 2015 Wiley Periodicals, Inc. Biopolymers 103: 550–562, 2015.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/112240/1/bip22657.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/112240/2/bip22657-sup-0004-suppinfo04.pd

Short-Range Imbalances in the AMBER Lennard-Jones Potential for (Deoxy)Ribose…Nucleobase Lone-pair…π Contacts in Nucleic Acids.

The lone-pair…π (lp…π) (deoxy)ribose…nucleobase stacking is a recurring structural motif in Z DNA and RNAs that is characterized by sub-van der Waals lp…π contacts (<3.0 Å). It is part of the structural signature of the CpG Z-steps in Z-DNA and r(UNCG) tetraloops. These nucleic acid structures are poorly behaving in molecular dynamics (MD) simulations. Although the exact origin of these issues remains unclear, a significant part of the problem might be due to an imbalanced description of non-bonded interactions including the characteristic lp…π stacking. To gain insights into the links between lp…π stacking and MD issues, we present an in-depth comparison between accurate large-basis-set double-hybrid Kohn-Sham density functional theory calculations DSD-BLYP-D3/ma-def2-QZVPP (DHDF-D3) and data obtained with the non-bonded potential of the AMBER force field (AFF) for NpN Z-steps (N = G, A, C, U). Among other differences, we found that the AFF overestimates the DHDF D3 lp…π distances by ~0.1-0.2 Å while the deviation between the DHDF-D3 and AFF descriptions sharply increases in the short-range region of the interaction. Based on atom-in-molecule (AIM) polarizabilities and SAPT analysis, we inferred that the DHDF-D3 vs. AFF differences partly originate in the Lennard-Jones (LJ) parameters that are identical for nucleobase carbon atoms despite the presence/absence of connected electron withdrawing groups that lead to different effective volumes or vdW radii. Thus, to precisely model the very short CpG lp…π contact distances, we recommend revision of the nucleobase atom LJ parameters. Additionally, we suggest that the large discrepancy between DHDF-D3 and AFF short-range repulsive part of the interaction energy potential may significantly contribute to the poor performances of MD simulations of nucleic acid systems containing Z-steps. Understanding where, and if possible why, the point-charge-type effective potentials reach their limits is vital for developing next-generation FFs and for addressing specific issues in contemporary MD simulations

High-speed narrow-bore capillary GC in combination with orthogonal accelerated time-of-flight mass spectrometric detection

A high-speed gas chromatographic system has been coupled to an orthogonal acceleration time-offlight mass analyzer. With this mass analyzer, it was possible to record a large number of transients per second. This instrument has also the ability of high resolution MS (up to 4(00), extremely high mass accuracy and produce spectra which shows good agreement with library spectra. The possibilities of this mass analyzer are discussed in detail and illustrated by an high-speed GC separation

High-speed narrow-bore capillary GC in combination with orthogonal accelerated time-of-flight mass spectrometric detection

A high-speed gas chromatographic system has been coupled to an orthogonal acceleration time-offlight mass analyzer. With this mass analyzer, it was possible to record a large number of transients per second. This instrument has also the ability of high resolution MS (up to 4(00), extremely high mass accuracy and produce spectra which shows good agreement with library spectra. The possibilities of this mass analyzer are discussed in detail and illustrated by an high-speed GC separation