Intracellular siRNA and precursor miRNA trafficking using bioresponsive copolypeptides.
BACKGROUND
Small interfering RNAs (siRNAs) can induce specific gene silencing through cytoplasmic mRNA cleavage and nuclear transcriptional silencing, necessitating delivery to different cellular compartments. This study presents a reducible copolypeptide (rCPP) carrier containing different molar ratios of a histidine-rich peptide (HRP) and nuclear localization sequence (NLS) peptide to modulate intracellular trafficking of transfected siRNA and primary RNA transcripts (pri-miRNA).


METHODS
Polyplex formation using siRNA and rCPP was demonstrated using photon correlation spectroscopy and atomic force microscopy. Confocal and fluorescence microscopy were used to investigate cellular uptake and nuclear trafficking whilst endogenous enhanced green fluorescent protein (EGFP) knockdown in H1299 cells was evaluated using flow cytometry. Transcriptional gene silencing of endogenous EF1A was verified using real-time reverse-transcription polymerase chain reaction (RT-PCR) and pri-miRNA nuclear processing was demonstrated using Northern analysis.


RESULTS
rCPP-based polyplexes showed rapid cellular uptake and low cytotoxicity. Labelled components revealed intact polyplexes after 2 h that exhibited directed movements consistent with endosomal trafficking. Polyplex-mediated knockdown of EGFP increased with greater HRP content. The inclusion of NLS promoted nuclear localization of transfected siRNAs and pri-miRNAs to the nuclear compartment allowing for transcriptional silencing of EF1A and Drosha and Dicer dependent expression of mature miRNA, respectively.


CONCLUSIONS
Our results demonstrate that reducible copolypeptides can be used as carriers for the non-toxic cellular delivery of siRNA and pri-miRNA. The nuclear targeting of rCPPs can be utilized for delivery of siRNAs and pri-miRNAs to the nuclear compartment for transcriptional gene silencing or endogenous processing into mature miRNA, respectively, which could potentially lead to improved therapeutic approaches.
Rahbek
Ulrik L
UL
Department of Molecular Biology, University of Aarhus, DK-8000, Denmark.
Howard
Kenneth A
KA
Oupicky
David
D
Manickam
Devika S
DS
Dong
Mingdong
M
Nielsen
Anne F
AF
Hansen
Thomas B
TB
Besenbacher
Flemming
F
Kjems
Jørgen
J
D002451:Cell Compartmentation; D016923:Cell Death / Q000187:drug effects; D002467:Cell Nucleus / Q000187:drug effects / Q000378:metabolism; D004229:Dithiothreitol / Q000494:pharmacology; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D006367:HeLa Cells; D006801:Humans; D035683:MicroRNAs / Q000235:genetics / Q000378:metabolism*; D008970:Molecular Weight; D010084:Oxidation-Reduction / Q000187:drug effects; D010455:Peptides / Q000737:chemistry / Q000378:metabolism*; D034622:RNA Interference / Q000187:drug effects; D012322:RNA Precursors / Q000378:metabolism*; D012323:RNA Processing, Post-Transcriptional / Q000187:drug effects; D034443:RNA Transport / Q000187:drug effects; D034741:RNA, Small Interfering / Q000378:metabolism*
D035683:MicroRNAs; D010455:Peptides; D012322:RNA Precursors; D034741:RNA, Small Interfering; D004229:Dithiothreitol