Ultrasensitive monitoring of ribozyme cleavage product using molecular-beacon-ligation system

Abstract

This paper reports a new approach to detect ribozyme cleavage product based on the molecularbeacon-ligation system. The molecular beacon, designed in such a way that one-half of its loop is complementary to ribozyme cleavage product, is used to monitor ligation process of RNA/DNA complex in a homogeneous solution and to convert directly cleavage product information into fluorescence signal. The method need not label ribozyme and ribozyme substrate, which is fast, simple and ultrasensitive for detection of cleavage product. Detection limit of the assay is 0.05 nmol/L. The cleavage product of hammerhead ribozyme against hepatitis C virus RNA (HCV-RNA) was detected perfectly based on this assay. Owing to its ultrasensitivity, excellent specificity, convenience and fidelity, this method might hold out great promise in ribozyme reaction and ribozyme gene therapy. molecular-beacon-ligation system, ribozyme cleavage, nucleic acids ligation, HCV ribozymeRibozyme is a catalytic RNA motif that cleaves the specific viral RNA and mRNA and results in the inhibition of gene expression [1] . Targeted destruction of viral RNA or cellular mRNA eliminates the formation of a protein that is deleterious in a disease state, which has potential therapeutic utility [2][3][4][5] . Ribozyme cleaves RNA substrate, resulting in a 2′,3′-cyclic phosphate product and a 5′-hydroxyl terminus product [6] . Traditionally, the cleavage process is usually assayed by radial labeling, denaturing gel electrophoresis and autoradiography [6 -9] . These methods are time consuming and complex. Recently, efforts have been made to develop fluorescence assays for ribozyme cleavage, including fluorescence resonance energy transfer [10][11][12] , fluorescent-labeled base-specific quenching [13] , fluorescence polarization [14] , 2-aminopurine probes [15] and ribozyme probes [16] . These methods perfectly monitored ribozyme reaction. However, these approaches need label ribozyme or substrates, which causes great limits in clinic applications.Molecular-beacon-ligation system was used to monitor DNA/DNA ligation reaction [17][18][19] and nucleic acids phosphorylation [20] in our laboratory. Here a new assay has been constructed to detect ribozyme cleavage product using molecular-beacon-ligation system without labeling procedure of ribozyme and substrate. The detection limit of the method is 0.05 nmol/L. In the range from 0.25 to 40 nmol/L, the initial ligation rate is directly proportional to the concentration of cleavage product. Major advantages of this method are its simplicity, ultrasensitivity, excellent specificity and high fidelity. The method will be widely useful for studying ribozyme cleavage process, ribozyme activity and relative gene therapy. Using this method, an efficient bioassay for the cleavage product of hammerhead ribozyme against hepatitis C virus RNA (HCV-RNA) has been developed.

Molecular Beacon Based Bioassay for Highly Sensitive and Selective Detection of Nicotinamide Adenine Dinucleotide and the Activity of Alanine Aminotransferase

“…[25][26][27] Recently, our group has utilized the advantages of MBs and various enzymes to develop a series of methods for real-time monitoring of nucleic acid ligation, 28 phosphorylation/dephosphorylation, 29,30 and double-stranded DNA cleavage. 31,32 Moreover, novel detection methods for DNA ligase, 33 polymerase, 34 restriction endonuclease, 35 and adenosine triphosphate (ATP) 36,37 have been developed. Herein, we developed a convenient, fast, highly sensitive, and selective assay scheme for the detection of NAD þ based on MB.…”

“…Since they were first introduced, molecular beacons (MBs) have been broadly used in the fields of biochemistry, biology, biotechnology, and medical research for their excellent sensitivity and specificity. − MBs can be not only a robust tool for DNA/RNA studies but also promising probes for monitoring DNA−protein interactions. − Recently, our group has utilized the advantages of MBs and various enzymes to develop a series of methods for real-time monitoring of nucleic acid ligation, phosphorylation/dephosphorylation, , and double-stranded DNA cleavage. , Moreover, novel detection methods for DNA ligase, polymerase, restriction endonuclease, and adenosine triphosphate (ATP) , have been developed.…”