A capillary electrophoretic assay for ribonuclease H activity

Chan, King C.; Budihas, Scott R.; Grice, Stuart F. J. Le; Parniak, Michael A.; Crouch, Robert J.; Gaidamakov, Sergei; Isaaq, Haleem J.; Wamiru, Antony; McMahon, James B.; Beutler, John A.

doi:10.1016/j.ab.2004.05.017

Cited by 27 publications

(21 citation statements)

References 24 publications

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“…Traditional techniques, including gel electrophoresis, high-performance liquid chromatography (HPLC), electrochemical studies, and the acid-soluble release of RNA fragments have been established in assays of enzyme activities and evaluations of the kinetic parameters. [11][12][13][14] These protocols share the drawbacks of being time-intensive, discontinuous, and laborious, and usually require isotope labeling. Many of these limitations are now being addressed by the development of fluorescence assays based on resonance energy transfer (FRET) or an excimer mechanism.…”

Section: Introductionmentioning

confidence: 99%

A Quadruplex‐Based, Label‐Free, and Real‐Time Fluorescence Assay for RNase H Activity and Inhibition

Huang

et al. 2010

Chemistry A European J

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We demonstrate a unique quadruplex-based fluorescence assay for sensitive, facile, real-time, and label-free detection of RNase H activity and inhibition by using a G-quadruplex formation strategy. In our approach, a RNA-DNA substrate was prepared, with the DNA strand designed as a quadruplex-forming oligomer. Upon cleavage of the RNA strand by RNase H, the released G-rich DNA strand folds into a quadruplex in the presence of monovalent ions and interacts with a specific G-quadruplex binder, N-methyl mesoporphyrin IX (NMM); this gives a dramatic increase in fluorescence and serves as a reporter of the reaction. This novel assay is simple in design, fast in operation, and is more convenient and promising than other methods. It takes less than 30 min to finish and the detection limit is much better or at least comparable to previous reports. No sophisticated experimental techniques or chemical modification for either RNA or DNA are required. The assay can be accomplished by using a common spectrophotometer and obviates possible interference with the kinetic behavior of the catalysts. Our approach offers an ideal system for high-throughput screening of enzyme inhibitors and demonstrates that the structure of the G-quadruplex can be used as a functional tool in specific fields in the future.

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Section: Introductionmentioning

confidence: 99%

A Quadruplex‐Based, Label‐Free, and Real‐Time Fluorescence Assay for RNase H Activity and Inhibition

Huang

et al. 2010

Chemistry A European J

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“…Attaching nitro group, methoxy-ether, or tertbutoxy-ester exhibited slight changes in compound potency (18)(19)(20). Extension of alkyl chain caused no significant difference in inhibitory potency (21). However, addition of hydroxy group decreased inhibitory activity (22).…”

Section: Resultsmentioning

confidence: 99%

“…Since a hydroxy group bound to phenyl ring is more reactive than a hydroxy group bound to alkane, the substitution reaction dominantly produced phenylester linkage (9-16) when a nucleophilic reagent contained two hydroxy groups. When a nucleophilic reagent contained only one hydroxy group, the substitution reaction generated alkyl-ester linkage (17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27). Compounds 28-30 were produced by generating nitro-furan-carbonyl-alkyl-benzene through the reaction of Weinreb amides containing benzyl group with alkyl lithium, followed by incorporation of nitro group into the phenyl ring using white fuming nitric acid and acetic anhydride.…”

Section: Methodsmentioning

confidence: 99%

“…A real-time monitoring assay was employed to estimate the IC 50 . 21,22) In short, two oligo-nucleotides were annealed at final concentrations of 2.5 and 0.25 µM for substrate. One was oligo-ribonucleotide 5′-GAUCUGAGCCUGGGAGCU-3′ with 6-carboxy-fluoroscein (FAM) conjugated at the 3′ end, and the other was oligodeoxyribonucleotide 5′-AGC TCC CAG GCT CAG ATC-3′ with black hole quencher (BHQ) conjugated at the 5′ end.…”

Section: Methodsmentioning

confidence: 99%

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Structural Modulation Study of Inhibitory Compounds for Ribonuclease H Activity of Human Immunodeficiency Virus Type 1 Reverse Transcriptase

Yanagita

Fudo

Urano

et al. 2012

CHEMICAL & PHARMACEUTICAL BULLETIN

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Reverse transcriptase of human immunodeficiency virus type 1 (HIV-1) has two enzymatic functions. One of the functions is ribonuclease (RNase) H activity concerning the digestion of only RNA of RNA/DNA hybrid. The RNase H activity is an attractive target for a new class of anti-HIV drugs because no approved inhibitor is available now. In our previous studies, an agent bearing 5-nitro-furan-2-carboxylic acid ester core was found from chemical screening and dozens of the derivatives were synthesized to improve compound potency. In this work, some parts of the chemical structure were modulated to deepen our understanding of the structure-activity relationship of the analogous compounds. Several derivatives having nitro-furan-phenyl-ester skeleton were shown to be potent RNase H inhibitors. Attaching methoxy-carbonyl and methoxy groups to the phenyl ring increased the inhibitory potency. No significant cytotoxicity was observed for these active derivatives. In contrast, the derivatives having nitro-furan-benzyl-ester skeleton showed modest inhibitory activities regardless of attaching diverse kinds of functional groups to the benzyl ring. Both the modulation of the 5-nitro-furan-2-carboxylic moiety and the conversion of the ester linkage resulted in a drastic decrease in inhibitory potency. These findings are informative for designing potent inhibitors of RNase H enzymatic activity of HIV-1.

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“…Enzymatic activity is then monitored with a colorimetric reaction with anti-digoxin antibody-alkaline phosphatase conjugate [8]. More recent assays use Xuorescein-labeled RNA oligonucleotides annealed to complementary DNA strands, and the enzymatic reactions are monitored with a spectroXuorometer [9,10] or by capillary electrophoresis [11].…”

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confidence: 99%