Live cell imaging of duplex siRNA intracellular trafficking

Autoren

Markus Hirsch
Mark Helm

DOI

10.1093/nar/gkv307

eISSN

1362-4962

ISSN

0305-1048

Ausgabe der Veröffentlichung

9

Zeitschrift

Nucleic Acids Research

Sprache

en

Online publication date

2015

Paginierung

4650 - 4660

Datum der Veröffentlichung

2015

Status

Published

Herausgeber

Oxford University Press (OUP)

Herausgeber URL

http://dx.doi.org/10.1093/nar/gkv307

Datum der Datenerfassung

2018

Titel

Live cell imaging of duplex siRNA intracellular trafficking

Ausgabe der Zeitschrift

43

Data source: Crossref

Abstract

Intracellular distribution of siRNA after in vitro transfection typically depends on lipopolyplexes, which must release the siRNA into the cytosol. Here, the fate of siRNAs was monitored by FRET-based live cell imaging. Subsequent to in situ observation of uptake and release processes, this approach allowed the observation of a number of hitherto uncharacterized intracellular distribution and degradation processes, commencing with a burst of endosomal releases, followed, in some cases, by fast siRNA influx into the nucleus. The continued observation of intact siRNA against a background of free fluorophores resulting from advanced degradation was possible by a specifically developed imaging algorithm, which identified populations of intact siRNA in pixels based on FRET. This proved to be essential in the end point definition of siRNA distribution, which typically featured partially degraded siRNA pools in perinuclear structures. Our results depict the initial 4 h as a critical time window, characterized by fast initial burst release into the cytosol, which lay the foundations for subsequent intracellular distribution of siRNA. Combination with a subsequent slower, but sustained release from endosomal reservoirs may contribute to the efficiency and duration of RNAi, and explain the success of lipopolyplexes in RNAi experiments in cell culture.

Addresses

Institute of Pharmacy and Biochemistry, University of Mainz, D-55128 Mainz, Germany Institute of Pharmacy und Molecular Biotechnology, University of Heidelberg, D-69120 Heidelberg, Germany.

Autoren

Markus Hirsch
Mark Helm

DOI

10.1093/nar/gkv307

eISSN

1362-4962

Externe Identifier

PubMed Identifier: 25870407
PubMed Central ID: PMC4482072

Open access

true

ISSN

0305-1048

Ausgabe der Veröffentlichung

9

Zeitschrift

Nucleic acids research

Schlüsselwörter

Cell Line
Cell Nucleus
Endosomes
Animals
Rats
RNA, Small Interfering
Microscopy, Confocal
Fluorescence Resonance Energy Transfer
Transfection
RNA Transport

Sprache

eng

Medium

Print-Electronic

Online publication date

2015

Open access status

Open Access

Paginierung

4650 - 4660

Datum der Veröffentlichung

2015

Status

Published

Publisher licence

CC BY

Datum der Datenerfassung

2015

Titel

Live cell imaging of duplex siRNA intracellular trafficking.

Sub types

Research Support, Non-U.S. Gov't
research-article
Journal Article

Ausgabe der Zeitschrift

43

Files

https://academic.oup.com/nar/article-pdf/43/9/4650/16661349/gkv307.pdf https://europepmc.org/articles/PMC4482072?pdf=render

Data source: Europe PubMed Central

Abstract

Intracellular distribution of siRNA after in vitro transfection typically depends on lipopolyplexes, which must release the siRNA into the cytosol. Here, the fate of siRNAs was monitored by FRET-based live cell imaging. Subsequent to in situ observation of uptake and release processes, this approach allowed the observation of a number of hitherto uncharacterized intracellular distribution and degradation processes, commencing with a burst of endosomal releases, followed, in some cases, by fast siRNA influx into the nucleus. The continued observation of intact siRNA against a background of free fluorophores resulting from advanced degradation was possible by a specifically developed imaging algorithm, which identified populations of intact siRNA in pixels based on FRET. This proved to be essential in the end point definition of siRNA distribution, which typically featured partially degraded siRNA pools in perinuclear structures. Our results depict the initial 4 h as a critical time window, characterized by fast initial burst release into the cytosol, which lay the foundations for subsequent intracellular distribution of siRNA. Combination with a subsequent slower, but sustained release from endosomal reservoirs may contribute to the efficiency and duration of RNAi, and explain the success of lipopolyplexes in RNAi experiments in cell culture.

Date of acceptance

2015

Autoren

Markus Hirsch
Mark Helm

Autoren-URL

https://www.ncbi.nlm.nih.gov/pubmed/25870407

DOI

10.1093/nar/gkv307

eISSN

1362-4962

Externe Identifier

PubMed Central ID: PMC4482072

Ausgabe der Veröffentlichung

9

Zeitschrift

Nucleic Acids Res

Schlüsselwörter

Animals
Cell Line
Cell Nucleus
Endosomes
Fluorescence Resonance Energy Transfer
Microscopy, Confocal
RNA Transport
RNA, Small Interfering
Rats
Transfection

Sprache

eng

Country

England

Paginierung

4650 - 4660

PII

gkv307

Datum der Veröffentlichung

2015

Status

Published

Datum, an dem der Datensatz öffentlich gemacht wurde

2015

Titel

Live cell imaging of duplex siRNA intracellular trafficking.

Sub types

Journal Article
Research Support, Non-U.S. Gov't

Ausgabe der Zeitschrift

43

Data source: PubMed

Live cell imaging of duplex siRNA intracellular trafficking

Files

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