Chimeric RNA-DNA molecular beacon assay for ribonuclease H activity.
Current methods to detect and assay ribonuclease H (RNase H) activity are indirect and time-consuming. Here we introduce a direct and sensitive method, based on the fluorescence quenching mechanism of molecular beacons, to assay RNA cleavage in RNA:DNA hybrids. An RNA-DNA chimeric beacon assay for RNase H enzymatic activity was developed. The substrate is a single-stranded RNA-DNA chimeric oligonucleotide labeled with a 5'-fluorescein and a 3'-DABCYL. The fluorophore (fluorescein) of the probe is held in close proximity to the quencher (DABCYL) by the RNA:DNA stem-loop structure. When the RNA sequence of the RNA:DNA hybrid stem is cleaved, the fluorophore is separated from the quencher and fluorescence can be detected as a function of time. Chimeric beacons with different stem lengths and sequences have been surveyed for this assay with E. coli RNase H. We found that the beacon kinetic parameters are in qualitative agreement with previously reported values using more cumbersome assays. This method permits real-time detection of RNase H activity and a convenient approach to RNase H kinetic and mechanistic study.
Rizzo
J
J
Department of Chemistry, School of Arts and Sciences, University of Pennsylvania, Philadelphia, PA, 19104, USA.
Gifford
L K
LK
Zhang
X
X
Gewirtz
A M
AM
Lu
P
P
D004247:DNA / Q000737:chemistry; D029968:Escherichia coli Proteins; D019793:Fluorescein; D005456:Fluorescent Dyes; D007700:Kinetics; D009690:Nucleic Acid Conformation; D015345:Oligonucleotide Probes / Q000138:chemical synthesis* / Q000737:chemistry / Q000378:metabolism; D012313:RNA / Q000737:chemistry; D016914:Ribonuclease H / Q000032:analysis* / Q000378:metabolism; D012680:Sensitivity and Specificity; D013050:Spectrometry, Fluorescence; D013329:Structure-Activity Relationship; D004124:p-Dimethylaminoazobenzene / Q000031:analogs & derivatives*
D029968:Escherichia coli Proteins; D005456:Fluorescent Dyes; D015345:Oligonucleotide Probes; C063127:4-(4-dimethylaminophenylazo)benzoic acid; D012313:RNA; D004247:DNA; D004124:p-Dimethylaminoazobenzene; D016914:Ribonuclease H; D019793:Fluorescein