Light‐Switching Excimer Beacon Assays For Ribonuclease H Kinetic Study

Abstract

RNase H is a ribonuclease that degrades the RNA strand in a RNA-DNA hybrid to produce 3'-hydroxyl-and 5'-phosphateterminated products. It is a nonspecific endonuclease that catalyzes the cleavage of RNA through an endonucleolytic mechanism, [1] aided by an enzyme-bound divalent metal ion; however, DNA strands or unhybridized RNA strands are not degraded. The enzyme is involved in several important cellular processes including DNA replication, DNA repair, and transcription. [2] Members of the RNase H family can be found in nearly all organisms, from archaea and prokaryota to eukaryota. RNase H also has wide applications in molecular biology and biotechnology in terms of its unique cleaving property. Retroviral RNase H, a part of viral reverse transcriptase, is an important pharmaceutical target, as it is absolutely necessary for the proliferation of retroviruses, such as HIV. Inhibitors of this enzyme could therefore provide new drugs against diseases like AIDS. E. coli RNase H usually requires at least six base pairs of RNA-DNA hybrids as substrates to bind and cleave effectively in solution, while the hybrid length required in living cells might be somewhat greater.[3] The complete digestion of poly(rA):poly(dT) by E. coli RNase H yields oligoribonucleotides with varying chain lengths, ranging from monomers to hexamers.[4] In order to understand more about these functions and processes, and more importantly, to screen new drugs against retroviruses, it is necessary to develop a fast, real-time, sensitive, and isotope-label-free system to assay the cleavage activity of RNase H. A number of traditional methods have been used to assay the enzyme activities and evaluate the kinetic parameters, such as the acid-soluble release of RNA fragment, [5] gel electrophoresis, [4] and HPLC.[6] The acid-soluble and gel-electrophoresis techniques require radioisotope-labeled substrates, and the HPLC method needs micromolar concentrations of substrate. All of these methods are indirect, discontinuous, and time-consuming. Recently, an RNA-DNA duplex was incorporated into a fluorescent probe to study RNase H in real time; however, only very short substrate sequences could be analyzed. Moreover, the complexity in the RNA-DNA oligonucleotide synthesis can also result in a low yield and restrict its general utility in enzyme-activity studies. [7] Here we describe a real-time fluorescence method in which the signal transduction is achieved by taking advantage of the light-switching excimer mechanism inherent to molecular beacons (MBs). A molecular beacon is a single-stranded DNA that can form an intramolecular hairpin structure with a fluorophore and quencher at either end.[8] DNA MB assays have been described for a few enzyme studies, such as single-stranded specific DNases, endonuclease BamHI, and small nonenzyme DNA cleavage agents.[9-11] Enzyme activity was detected and characterized by taking advantage of the signal-transduction mechanism built into the MBs. The change in fluorescence signal reflects the conformational change of...

Telomeric repeat-containing RNA structure in living cells

“…1) (16), however, demonstration of their formation in living cells remains elusive. To investigate whether TERRA RNA G-quadruplexes exist in living cells, we employed a light-switching pyrene probe that has been designed to employ the capability of pyrene to form fluorescent excited-state dimers (excimers) (25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35)(36). The advantage of the distance dependence of excimer formation with pyrene can be used as a unique excimer signaling device for detecting G-quadruplex structure ( Fig.…”

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

“…Many of these limitations are now being addressed by the development of fluorescence assays based on resonance energy transfer (FRET) or an excimer mechanism. [2,[15][16][17] Although promising, these techniques are compromised by the requirement for double-labeled oligonucleotide substrates, limited chemical stability, and interference by external nonspecific events. In addition, the possibility that the bulky fluorescent groups might interfere with the kinetic behavior of the catalysts is always a concern.…”

“…K M for RNase H has been reported to vary from 19 to 4.2 mm. [2,12,17,29] The variation is the result of a variety of assay conditions, substrates, and methods.…”

“…[17] Furthermore, more sensitive detection could be achieved by optimizing the G-quadruplexforming sequence and the cleavage conditions, for example, increasing the concentration of substrates or prolonging the cleavage time.…”

Synthesis and Optical Properties of Cyanine Dyes as Fluorescent DNA Base Substitutions for Live Cell Imaging

“…DNA with intrastrand and interstrand dimers mainly of pyrene and perylene exhibit strong excimer fluorescence [11][12][13][14][15] and can also be applied in molecular beacons. [16][17][18][19] However, the excitation of pyrene requires highenergy UV light, a circumstance that limits the bioanalytic and imaging applications significantly. Recently, we reported that the excitation of perylene bisimide pairs at 505 nm yields an excimer-type shift of the fluorescence by ca.…”