Single-molecule detection and tracking of RNA transcripts in living cells using phosphorothioate-optimized 2'-O-methyl RNA molecular beacons.
Molecular Beacons (MBs) composed of 2'-O-methyl RNA (2Me) and phosphorothioate (PS) linkages throughout the backbone (2Me/PSFULL MBs) have enabled long-term imaging of RNA in living cells, but excess PS modification can induce nonspecific binding, causing false-positive signals. In this study, we evaluate the intracellular stability of MBs composed of 2Me with various PS modifications, and found that false-positive signals could be reduced to marginal levels when the MBs possess a fully PS-modified loop domain and a phosphodiester stem (2Me/PSLOOP MB). Additionally, 2Me/PSLOOP MBs exhibited uncompromised hybridization kinetics, prolonged functionality and >88% detection accuracy for single RNA transcripts, and could do so without interfering with gene expression or cell growth. Finally, 2Me/PSLOOP MBs could image the dynamics of single mRNA transcripts in the nucleus and the cytoplasm simultaneously, regardless of whether the MBs targeted the 5'- or the 3'-UTR. Together, these findings demonstrate the effectiveness of loop-domain PS modification in reducing nonspecific signals and the potential for sensitive and accurate imaging of individual RNAs at the single-molecule level. With the growing interest in the role of RNA localization and dynamics in health and disease, 2Me/PSLOOP MBs could enable new discoveries in RNA research.
Department of Biomedical Engineering, College of Engineering, Peking University, Beijing, 100871, China.
Dan
D
Zhao
Department of Biomedical Engineering, College of Engineering, Peking University, Beijing, 100871, China.
Yantao
Y
Yang
Department of Biomedical Engineering, College of Engineering, Peking University, Beijing, 100871, China.
Na
N
Qu
Department of Biomedical Engineering, College of Engineering, Peking University, Beijing, 100871, China.
Mingming
M
Chen
Department of Biomedical Engineering, College of Engineering, Peking University, Beijing, 100871, China.
Zhao
Z
Ma
Department of Biomedical Engineering, College of Engineering, Peking University, Beijing, 100871, China.
Christopher J
CJ
Krueger
Integrated DNA Technologies Inc., Coralville, IA, 52241, USA.
Mark A
MA
Behlke
Department of Biomedical Engineering, College of Engineering, Peking University, Beijing, 100871, China. Electronic address: chenak@pku.edu.cn.
Antony K
AK
Chen
D002460:Cell Line; D005456:Fluorescent Dyes / Q000737:chemistry*; D049452:Green Fluorescent Proteins / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D008745:Methylation; D008856:Microscopy, Fluorescence; D009693:Nucleic Acid Hybridization; D015345:Oligonucleotide Probes / Q000737:chemistry* / Q000235:genetics; D054735:Phosphorothioate Oligonucleotides / Q000737:chemistry* / Q000235:genetics; D012333:RNA, Messenger / Q000032:analysis* / Q000235:genetics; D013050:Spectrometry, Fluorescence; D014158:Transcription, Genetic
D005456:Fluorescent Dyes; D015345:Oligonucleotide Probes; D054735:Phosphorothioate Oligonucleotides; D012333:RNA, Messenger; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins
2′-O-methyl RNA; Molecular beacons; Phosphorothioate; RNA dynamics; RNA imaging; Single-molecule detection