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Probing fast ribozyme reactions under biological conditions with rapid quench-flow kinetics.

ABSTRACT: Reaction kinetics on the millisecond timescale pervade the protein and RNA fields. To study such reactions, investigators often perturb the system with abiological solution conditions or substrates in order to slow the rate to timescales accessible by hand mixing; however, such perturbations can change the rate-limiting step and obscure key folding and chemical steps that are found under biological conditions. Mechanical methods for collecting data on the millisecond timescale, which allow these perturbations to be avoided, have been developed over the last few decades. These methods are relatively simple and can be conducted on affordable and commercially available instruments. Here, we focus on using the rapid quench-flow technique to study the fast reaction kinetics of RNA enzymes, or ribozymes, which often react on the millisecond timescale under biological conditions. Rapid quench of ribozymes is completely parallel to the familiar hand-mixing approach, including the use of radiolabeled RNAs and fractionation of reactions on polyacrylamide gels. We provide tips on addressing and preventing common problems that can arise with the rapid-quench technique. Guidance is also offered on ensuring the ribozyme is properly folded and fast-reacting. We hope that this article will facilitate the broader use of rapid-quench instrumentation to study fast-reacting ribozymes under biological reaction conditions.

SUBMITTER: Bingaman JL

PROVIDER: S-EPMC5447473 | biostudies-literature | 2017 May

REPOSITORIES: biostudies-literature

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Probing fast ribozyme reactions under biological conditions with rapid quench-flow kinetics.

Bingaman Jamie L JL Messina Kyle J KJ Bevilacqua Philip C PC

Methods (San Diego, Calif.) 20170314

Reaction kinetics on the millisecond timescale pervade the protein and RNA fields. To study such reactions, investigators often perturb the system with abiological solution conditions or substrates in order to slow the rate to timescales accessible by hand mixing; however, such perturbations can change the rate-limiting step and obscure key folding and chemical steps that are found under biological conditions. Mechanical methods for collecting data on the millisecond timescale, which allow these ...[more]

PMID: 28315484

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Project description:Bioaerosols exposure can lead to many adverse health effects and even result in death if highly infectious agents involved. Apparently, there is a great need for rapid detection of bioaerosols, for which air sampling often is the first critical step. However, currently available samplers often either require an external power and/or with low sampling flow rate, thus falling short of providing a practical solution when response time is of great concern. Here, we have designed and evaluated a new portable high volume bioaerosol sampler named as HighBioTrap through optimizing its operating parameters. The sampler was operated at a sampling flow rate of 1200 L/min, with an impaction velocity of about 10.2 m/s (S/W = 1.5, T/W = 1), while the weight of the sampler is about 1.9 kg. The performances of the HighBioTrap sampler were tested both in lab controlled and natural environments (outdoor and indoor environments in a university building) along with the reference sampler-the BioStage impactor using aerosolized Polystyrene (PS) uniform microspheres of various sizes, aerosolized bacteria and also ambient air particles. The microbial community structures of collected culturable bacterial aerosol particles both by the HighBioTrap and the BioStage impactor in the natural environments were analyzed using gene sequence method. Experimental results with PS particles showed the HighBioTrap has a cutoff size of ~ 2 µm. The widely used impactor design equation was found to be not applicable for predicting the performance of the HighBioTrap due to its large Reynolds number. When sampling aerosolized individual Pseudomonas fluorescens and Bacillus subtilis bacterial particles, the HighBioTrap had physical collection efficiencies of 10% and 20%, respectively. Despite the higher desiccation effects introduced by higher flow rate, the HighBioTrap was shown to obtain a higher microbial diversity than the BioStage impactor for both in outdoor and indoor environments given the same sampling time (p < 0.01). Our data also showed that most of the desiccation effects might have occurred between 3 and 5 min of the sampling and an impaction velocity of around 10 m/s might be a close-to-optimal impaction velocity for collecting most environmental bacterial aerosols while maximally preserving their culturability. This work contributes to our understanding of microbial sampling stress (impaction velocity and sampling time), while developing a portable high volume sampler. The HighBioTrap sampler could find its great efficiencies in qualitative microbial aerosol detection and analysis, such as investigation of microbial aerosol diversity for a particular environment, or when the low level of pathogens is present and detection time is of great concern.

Dataset Information

Probing fast ribozyme reactions under biological conditions with rapid quench-flow kinetics.

Publications

Probing fast ribozyme reactions under biological conditions with rapid quench-flow kinetics.

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