Expedited quantification of mutant ribosomal RNA by binary deoxyribozyme (BiDz) sensors.
Mutations in ribosomal RNA (rRNA) have traditionally been detected by the primer extension assay, which is a tedious and multistage procedure. Here, we describe a simple and straightforward fluorescence assay based on binary deoxyribozyme (BiDz) sensors. The assay uses two short DNA oligonucleotides that hybridize specifically to adjacent fragments of rRNA, one of which contains a mutation site. This hybridization results in the formation of a deoxyribozyme catalytic core that produces the fluorescent signal and amplifies it due to multiple rounds of catalytic action. This assay enables us to expedite semi-quantification of mutant rRNA content in cell cultures starting from whole cells, which provides information useful for optimization of culture preparation prior to ribosome isolation. The method requires less than a microliter of a standard Escherichia coli cell culture and decreases analysis time from several days (for primer extension assay) to 1.5 h with hands-on time of ∼10 min. It is sensitive to single-nucleotide mutations. The new assay simplifies the preliminary analysis of RNA samples and cells in molecular biology and cloning experiments and is promising in other applications where fast detection/quantification of specific RNA is required.
Chemistry Department, University of Central Florida, Orlando, Florida 32816, USA.
Yulia V
YV
Gerasimova
Center for BioEnergetics, Biodesign Institute, Arizona State University, Tempe, Arizona 85287, USA Department of Chemistry and Biochemistry, Arizona State University, Tempe, Arizona 85287, USA.
Petro
P
Yakovchuk
Center for BioEnergetics, Biodesign Institute, Arizona State University, Tempe, Arizona 85287, USA Department of Chemistry and Biochemistry, Arizona State University, Tempe, Arizona 85287, USA.
Larisa M
LM
Dedkova
Center for BioEnergetics, Biodesign Institute, Arizona State University, Tempe, Arizona 85287, USA Department of Chemistry and Biochemistry, Arizona State University, Tempe, Arizona 85287, USA.
Sidney M
SM
Hecht
Chemistry Department, University of Central Florida, Orlando, Florida 32816, USA National Center for Forensic Science, University of Central Florida, Orlando, Florida 32816, USA Burnett School of Biomedical Sciences, University of Central Florida, Orlando, Florida 32816, USA.
Dmitry M
DM
Kolpashchikov
D001483:Base Sequence; D015374:Biosensing Techniques; D021881:DNA, Catalytic / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D008969:Molecular Sequence Data; D009154:Mutation; D009690:Nucleic Acid Conformation; D009693:Nucleic Acid Hybridization; D012329:RNA, Bacterial / Q000737:chemistry / Q000235:genetics*; D012335:RNA, Ribosomal / Q000737:chemistry / Q000235:genetics*
D021881:DNA, Catalytic; D012329:RNA, Bacterial; D012335:RNA, Ribosomal
binary deoxyribozyme; fluorescent sensors; mix-and-read probes; mutation analysis; ribosomal RNA