Thermodynamic examination of pH and magnesium effect on U6 RNA internal loop

OˈConnell, Allison; Hanson, Jared A.; McCaskill, Darryl C.; Moore, Ethan; Lewis, Daniel C.; Grover, Neena

doi:10.1261/rna.070466.119

Cited by 6 publications

(3 citation statements)

References 53 publications

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“…When evaluating EhhRzs under single-turnover conditions (10-fold molar excess of enzyme over substrate [at 1 μM]), pre-annealed and initiated by 0.5 mM Mg 2+ , we were unable to measure the rate with current optical methods of the A7U(AGUA) enzyme because of the rapid speed (estimated >1000 nM min -1 ), we can only estimate the A7U(GAAA) enzyme at >680 nM min −1 and obtain a rate of approximately 273 nM min −1 for the WT(GAAA) enzyme. We did not explore reduced pH, because the optical assay designed here depends upon product release, and base pairing can be affected by pH (Thapylal et al, 2014; Szabat and Kierzek, 2017; O’Connell et al, 2019). Rapid kinetic assays (stopped-flow) are needed to investigate these rates under physiological conditions (Bingaman et al, 2017).…”

Section: Discussionmentioning

confidence: 99%

Log-order improved in trans hammerhead ribozyme turnover rates: reevaluating therapeutic space for small catalytic RNAs

Myers

Sullivan

2022

Preprint

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We discovered an enhanced functionality hammerhead ribozyme (EhhRz), designed to act in trans against human rod opsin (RHO) mRNA, with turnover activity >300 nM min−1 under substrate-excess conditions and physiological Mg2+ levels (1 mM). We developed a real-time moderate-throughput fluorescence quantitative hhRz kinetic assay, which is linear with substrate and product moles and supported by gel-based measures. The EhhRz targets a CUC↓ cleavage site in a substrate with no predicted secondary/tertiary structure and demonstrates classic Michaelis-Menten turnover behavior when the substrate is in 10-fold excess (Vmax/Km up to 1.60 × 108 min−1 M−1), which is comparable to RNase A. EhhRzs show cooperative titration with a Kd of 0.73 ± 0.02 mM at cellular Mg2+ concentrations and a Hill coefficient of 1.73 ± 0.07. The upstream EhhRz antisense flank (bound to a downstream substrate flank) interacts with stem-loop II, and examinations of different variants revealed that a U7 residue in the downstream flank of the substrate is not essential for enhanced activity. Under single-turnover conditions with substrate pre-annealed to enzyme, reaction rates exceeded 1,000 min−1. These findings show that RNA catalysis approaches the efficiency of the ribosome and suggests EhhRz in trans is a druggable nucleic acid therapeutic.

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Section: Discussionmentioning

confidence: 99%

Log-order improved in trans hammerhead ribozyme turnover rates: reevaluating therapeutic space for small catalytic RNAs

Myers

Sullivan

2022

Preprint

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“…The mean age, postmortem interval (PMI), tissue storage time, and RNA integrity number (RIN) did not differ between subject groups (Table 1). Brain pH signi cantly differed between subject groups (t 19 = 2.2, p = 0.039), but the mean difference was 0.2 pH units and of uncertain biological relevance 31 .…”

Section: Human Subjectsmentioning

confidence: 95%

Altered Rbfox1-Vamp1 pathway and prefrontal cortical dysfunction in schizophrenia

Chung,

Dienel,

Belch

et al. 2024

Mol Psychiatry

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De cient gamma oscillations in prefrontal cortex (PFC) of individuals with schizophrenia appear to involve impaired inhibitory drive from parvalbumin-expressing interneurons (PVIs). Inhibitory drive from PVIs is regulated, in part, by RNA binding fox-1 homolog 1 (Rbfox1). Rbfox1 is spliced into nuclear or cytoplasmic isoforms, which regulate alternative splicing or stability of their target transcripts, respectively. One major target of cytoplasmic Rbfox1 is vesicle associated membrane protein 1 (Vamp1). Vamp1 mediates GABA release probability from PVIs, and the loss of Rbfox1 reduces Vamp1 levels which in turn impairs cortical inhibition. In this study, we investigated if the Rbfox1-Vamp1 pathway is altered in PVIs in PFC of individuals with schizophrenia by utilizing a novel strategy that combines multilabel in situ hybridization and immunohistochemistry. In the PFC of 20 matched pairs of schizophrenia and comparison subjects, cytoplasmic Rbfox1 protein levels were signi cantly lower in PVIs in schizophrenia and this de cit was not attributable to potential methodological confounds or schizophrenia-associated co-occurring factors. In a subset of this cohort, Vamp1 mRNA levels in PVIs were also signi cantly lower in schizophrenia and were predicted by lower cytoplasmic Rbfox1 protein levels across individual PVIs. To investigate the functional impact of Rbfox1-Vamp1 alterations in schizophrenia, we simulated the effect of lower GABA release probability from PVIs on gamma power in a computational model network of pyramidal neurons and PVIs. Our simulations showed that lower GABA release probability reduces gamma power by disrupting network synchrony while minimally affecting network activity. Finally, lower GABA release probability synergistically interacted with lower strength of inhibition from PVIs in schizophrenia to reduce gamma power non-linearly. Together, our ndings suggest that the Rbfox1-Vamp1 pathway in PVIs is impaired in schizophrenia and that this alteration likely contributes to de cient PFC gamma power in the illness.

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“…The significance of the protonation of nucleobases in biological macromolecules has been discussed in numerous publications ( 20–24 ). One of the most recent examples is the spliceosome ( 25 ), in which one of the base pairs formed between adenine and cytosine has to be protonated to facilitate the conformational change necessary for the spliceosome's assembly and catalysis.…”

Section: Introductionmentioning

confidence: 99%

Frequency and hydrogen bonding of nucleobase homopairs in small molecule crystals

Cabaj

Dominiak

2020

Nucleic Acids Research

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We used the high resolution and accuracy of the Cambridge Structural Database (CSD) to provide detailed information regarding base pairing interactions of selected nucleobases. We searched for base pairs in which nucleobases interact with each other through two or more hydrogen bonds and form more or less planar structures. The investigated compounds were either free forms or derivatives of adenine, guanine, hypoxanthine, thymine, uracil and cytosine. We divided our findings into categories including types of pairs, protonation patterns and whether they are formed by free bases or substituted ones. We found base pair types that are exclusive to small molecule crystal structures, some that can be found only in RNA containing crystal structures and many that are native to both environments. With a few exceptions, nucleobase protonation generally followed a standard pattern governed by pKa values. The lengths of hydrogen bonds did not depend on whether the nucleobases forming a base pair were charged or not. The reasons why particular nucleobases formed base pairs in a certain way varied significantly.

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Thermodynamic examination of pH and magnesium effect on U6 RNA internal loop

Cited by 6 publications

References 53 publications

Log-order improved in trans hammerhead ribozyme turnover rates: reevaluating therapeutic space for small catalytic RNAs

Log-order improved in trans hammerhead ribozyme turnover rates: reevaluating therapeutic space for small catalytic RNAs

Altered Rbfox1-Vamp1 pathway and prefrontal cortical dysfunction in schizophrenia

Frequency and hydrogen bonding of nucleobase homopairs in small molecule crystals

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