A novel sequence-specific RNA quantification method using nicking endonuclease, dual-labeled fluorescent DNA probe, and conformation-interchangeable oligo-DNA

Abstract

We have developed a novel, single-step, isothermal, signal-amplified, and sequence-specific RNA quantification method (Lassay). The L-assay consists of nicking endonuclease, a dual-labeled fluorescent DNA probe (DL-probe), and conformationinterchangeable oligo-DNA (L-DNA). This signal-amplified assay can quantify target RNA concentration in a sequence-specific manner with a coefficient of variation (Cv) of 5% and a lower limit of detection of 0.1 nM. Moreover, this assay allows quantification of target RNA even in the presence of a several thousandfold excess by weight of cellular RNA. In addition, this assay can be used to measure the changes in RNA concentration in real-time and to quantify short RNAs (<30 nucleotides). The L-assay requires only incubation under isothermal conditions, is inexpensive, and is expected to be useful for basic research requiring high-accuracy, easy-to-use RNA quantification, and real-time quantification.

Natural and engineered nicking endonucleases--from cleavage mechanism to engineering of strand-specificity

“…The opening of the probe exposes a sequence complementary to a quenched fluorescent probe with a 5′-fluorophore and a 3′-quencher. Annealing with the quenched probe generates a ds-BbvCI site such that Nb.BbvCI nicks the strand containing quenched fluorescent probe and releases the fluorophore from being quenched ( 114 ). This RNA detection scheme has been shown to be quantitative and not requiring purification of the RNA, and should be applicable to the detection of ss DNA.…”

Peculiarities of the interaction of the restriction endonuclease BspD6I with DNA containing its recognition site

“…NEs themselves nowadays are widely used, e.g. for DNA amplification [8], in sequencing procedures [9], for DNA [10] or RNA detection and quantification [11]. To improve the existing methods and to develop new approaches, it is necessary to acquire information about the mechanism of NE action.…”

An ultrahighly sensitive and selective electrochemical DNA sensor via nicking endonuclease assisted current change amplification

“…Recently, a novel nucleic acid detection technique called nicking endonuclease enhanced signal amplification (NESA) has been reported by Kiesling et al 12 As a new nucleic acid detection technique, NESA has been reported to have distinct specificity for the detection of a single-base mismatch. However, the sensitivity of previously reported NESA was not high enough (only in the picomolar level), even when molecular beacons, 13 a dual-labeled fluorescent DNA probe, 14 or nanoparticles 15 were used to increase the sensitivity. Furthermore, the NESA-based DNA detection methods previously reported have other limitations such as complexity of fabrication and are time consuming.…”