Peptide-linked molecular beacons for efficient delivery and rapid mRNA detection in living cells.
Real-time visualization of specific endogenous mRNA expression in vivo has the potential to revolutionize medical diagnosis, drug discovery, developmental and molecular biology. However, conventional liposome- or dendrimer-based cellular delivery of molecular probes is inefficient, slow, and often detrimental to the probes. Here we demonstrate the rapid and sensitive detection of RNA in living cells using peptide-linked molecular beacons that possess self-delivery, targeting and reporting functions. We conjugated the TAT peptide to molecular beacons using three different linkages and demonstrated that, at relatively low concentrations, these molecular beacon constructs were internalized into living cells within 30 min with nearly 100% efficiency. Further, peptide-based delivery did not interfere with either specific targeting by or hybridization-induced fluorescence of the probes. We could therefore detect human GAPDH and survivin mRNAs in living cells fluorescently, revealing intriguing intracellular localization patterns of mRNA. We clearly demonstrated that cellular delivery of molecular beacons using the peptide-based approach has far better performance compared with conventional transfection methods. The peptide-linked molecular beacons approach promises to open new and exciting opportunities in sensitive gene detection and quantification in vivo.
Nitin
Nitin
N
Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, GA 30332, USA.
Santangelo
Philip J
PJ
Kim
Gloria
G
Nie
Shuming
S
Bao
Gang
G
D000595:Amino Acid Sequence; D026921:Amino Acid Transport Systems, Neutral; D001483:Base Sequence; D002352:Carrier Proteins / Q000032:analysis / Q000737:chemistry / Q000378:metabolism*; D002460:Cell Line; D002470:Cell Survival; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000235:genetics; D006801:Humans; D017404:In Situ Hybridization, Fluorescence; D051034:Inhibitor of Apoptosis Proteins; D007700:Kinetics; D008869:Microtubule-Associated Proteins / Q000235:genetics; D015335:Molecular Probes / Q000032:analysis* / Q000235:genetics; D009363:Neoplasm Proteins; D009693:Nucleic Acid Hybridization; D010455:Peptides / Q000032:analysis / Q000737:chemistry / Q000378:metabolism*; D034443:RNA Transport; D012333:RNA, Messenger / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D012996:Solutions; D000077022:Survivin; D014162:Transfection
D026921:Amino Acid Transport Systems, Neutral; C107489:BIRC5 protein, human; D002352:Carrier Proteins; D051034:Inhibitor of Apoptosis Proteins; D008869:Microtubule-Associated Proteins; D015335:Molecular Probes; D009363:Neoplasm Proteins; D010455:Peptides; D012333:RNA, Messenger; C498964:SLC16A10 protein, human; D012996:Solutions; D000077022:Survivin; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases