Fast, Facile, Hypersensitive Assays for Ribonucleolytic Activity

Park, Chiwook; Kelemen, Bradley R.; Klink, Tony A.; Sweeney, Rozamond Y.; Behlke, Mark A.; Eubanks, Shad R.; Raines, Ronald T.

doi:10.1016/s0076-6879(01)41146-3

Cited by 33 publications

(51 citation statements)

References 34 publications

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“…The RNase A detection method previously reported [3][4][5][6]26 was 0.5 pg RNase A and the the detection limit of this method is in good agreement with that previously reported. These results showed that RNase could be detected by the mRNA-immobilized electrode coupled with 1.…”

Section: Treatment Of Mrna-immobilized Electrode With Rnase Asupporting

confidence: 88%

“…[3][4][5][6]26 This electrochemical detection method, based on decreasing current still remains to be improved. However, we could succeed in a highly sensitive electrochemical detection of RNase A, even under the conditions employed here.…”

Section: Discussionmentioning

confidence: 99%

“…So far, a hypersensitive fluorometric RNase detection method is known with the RNase-substrate oligonucleotide having fluorescein and rhodamine dyes at both termini. [3][4][5][6] This oligonucleotide substrate shows fluorescence quenching of the fluorescein by the proximal rhodamine in an aqueous solution. However, in the presence of RNase, the fluorescence of the fluorescein was recovered by cleavage of the oligonucleotide due to RNase digestion.…”

Section: Introductionmentioning

confidence: 99%

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Ferrocenylnaphthalene Diimide-based Electrochemical Ribonuclease Assay

et al. 2007

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Section: Treatment Of Mrna-immobilized Electrode With Rnase Asupporting

confidence: 88%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Ferrocenylnaphthalene Diimide-based Electrochemical Ribonuclease Assay

et al. 2007

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“…Fluorogenic substrates have previously been used to analyze the reaction kinetics of ribonucleases (Park et al 2001;Wang and Hergenrother 2007) and generally consist of single RNA bases flanked by short DNA sequences. These mixed oligonucleotides are labeled with a fluorophore at one end and a quencher at the other.…”

Section: Kinetic Analysis Of Vapcmentioning

confidence: 99%

Determination of ribonuclease sequence-specificity using Pentaprobes and mass spectrometry

McKenzie

Duyvestyn²,

Smith³

et al. 2012

RNA

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The VapBC toxin-antitoxin (TA) family is the largest of nine identified TA families. The toxin, VapC, is a metal-dependent ribonuclease that is inhibited by its cognate antitoxin, VapB. Although the VapBCs are the largest TA family, little is known about their biological roles. Here we describe a new general method for the overexpression and purification of toxic VapC proteins and subsequent determination of their RNase sequence-specificity. Functional VapC was isolated by expression of the nontoxic VapBC complex, followed by removal of the labile antitoxin (VapB) using limited trypsin digestion. We have then developed a sensitive and robust method for determining VapC ribonuclease sequence-specificity. This technique employs the use of Pentaprobes as substrates for VapC. These are RNA sequences encoding every combination of five bases. We combine the RNase reaction with MALDI-TOF MS to detect and analyze the cleavage products and thus determine the RNA cut sites. Successful MALDI-TOF MS analysis of RNA fragments is acutely dependent on sample preparation methods. The sequencespecificity of four VapC proteins from two different organisms (VapC PAE0151 and VapC PAE2754 from Pyrobaculum aerophilum, and VapC Rv0065 and VapC Rv0617 from Mycobacterium tuberculosis) was successfully determined using the described strategy. This rapid and sensitive method can be applied to determine the sequence-specificity of VapC ribonucleases along with other RNA interferases (such as MazF) from a range of organisms.

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“…Fluorogenic substrates have been used to analyze ribonuclease kinetics, substrate specificity [28], and inhibition [29][30][31]. These substrates are often chimeric, as they are composed of a single RNA base flanked by short (1 -4 bases) sequences of DNA bases, and they are labeled on one end with a fluorophore and on the other end with a quencher.…”

Section: Substrate Designmentioning

confidence: 99%

A continuous fluorometric assay for the assessment of MazF ribonuclease activity

Wang

Hergenrother

2007

Analytical Biochemistry

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Plasmids maintain themselves in their bacterial host through several different mechanisms, one of which involves the synthesis of plasmid-encoded toxin and antitoxin proteins. When the plasmid is present the antitoxin binds to and neutralizes the toxin. If a plasmid-free daughter cell arises, however, the labile antitoxin is degraded (and not replenished) and the toxin kills the cell from within. These toxin-antitoxin (TA) systems thereby function as post-segregational killing systems, and the disruption of the TA interaction represents an intriguing antibacterial strategy. It was recently discovered that the genes for one particular TA system, MazEF, are ubiquitous on plasmids isolated from clinical vancomycin-resistant enterococci (VRE) strains. It thus appears that small molecule disruptors of the MazEF interaction have potential as antibacterial agents. The MazF toxin protein is known to be a ribonuclease. Unfortunately, traditional methods for the assessment of MazF activity rely on the use of radiolabeled substrates followed by analysis with polyacrylamide gel electrophoresis. Described herein is a simple and convenient continuous assay for the assessment of MazF activity. This assay utilizes an oligonucleotide with a fluorophore on the 5′-end and a quencher on the 3′-end, and processing of this substrate by MazF results in a large increase in the fluorescence signal. Through this assay we have for the first time determined a K M and V max for this enzyme, and have also found that MazF is not inhibited by standard ribonuclease inhibitors. This assay will be useful to those interested in the biochemistry of the MazF family of toxins and the disruption of MazE-MazF.

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Fast, Facile, Hypersensitive Assays for Ribonucleolytic Activity

Cited by 33 publications

References 34 publications

Ferrocenylnaphthalene Diimide-based Electrochemical Ribonuclease Assay

Ferrocenylnaphthalene Diimide-based Electrochemical Ribonuclease Assay

Determination of ribonuclease sequence-specificity using Pentaprobes and mass spectrometry

A continuous fluorometric assay for the assessment of MazF ribonuclease activity

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