Quantification of functionalised gold nanoparticle-targeted knockdown of gene expression in HeLa cells

Meesbah Jiwaji, Mairi E. Sandison, Julien Reboud, Ross Stevenson, Rónán Daly, Gráinne Barkess, Karen Faulds, Walter Kolch, Duncan Graham, Mark A. Girolami, Jonathan M. Cooper, Andrew Pitt

Research output: Contribution to journalArticle

Abstract

Introduction: Gene therapy continues to grow as an important area of research, primarily because of its potential in the treatment of disease. One significant area where there is a need for better understanding is in improving the efficiency of oligonucleotide delivery to the cell and indeed, following delivery, the characterization of the effects on the cell. Methods: In this report, we compare different transfection reagents as delivery vehicles for gold nanoparticles functionalized with DNA oligonucleotides, and quantify their relative transfection efficiencies. The inhibitory properties of small interfering RNA (siRNA), single-stranded RNA (ssRNA) and single-stranded DNA (ssDNA) sequences targeted to human metallothionein hMT-IIa are also quantified in HeLa cells. Techniques used in this study include fluorescence and confocal microscopy, qPCR and Western analysis. Findings: We show that the use of transfection reagents does significantly increase nanoparticle transfection efficiencies. Furthermore, siRNA, ssRNA and ssDNA sequences all have comparable inhibitory properties to ssDNA sequences immobilized onto gold nanoparticles. We also show that functionalized gold nanoparticles can co-localize with autophagosomes and illustrate other factors that can affect data collection and interpretation when performing studies with functionalized nanoparticles. Conclusions: The desired outcome for biological knockdown studies is the efficient reduction of a specific target; which we demonstrate by using ssDNA inhibitory sequences targeted to human metallothionein IIa gene transcripts that result in the knockdown of both the mRNA transcript and the target protein.

LanguageEnglish
Article numbere99458
Number of pages10
JournalPLoS ONE
Volume9
Issue number6
DOIs
Publication statusPublished - 13 Jun 2014

Fingerprint

single-stranded DNA
transfection
HeLa Cells
Gene expression
Gold
Nanoparticles
Single-Stranded DNA
DNA sequences
Transfection
Gene Expression
gene expression
metallothionein
nanoparticles
small interfering RNA
oligonucleotides
nucleotide sequences
Metallothionein
Oligonucleotides
Small Interfering RNA
RNA

Bibliographical note

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (CC-BY), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Funding: EPSRC (Basic Technology Research Grant, grant number EP/E032745/1).

Cite this

Jiwaji, Meesbah ; Sandison, Mairi E. ; Reboud, Julien ; Stevenson, Ross ; Daly, Rónán ; Barkess, Gráinne ; Faulds, Karen ; Kolch, Walter ; Graham, Duncan ; Girolami, Mark A. ; Cooper, Jonathan M. ; Pitt, Andrew. / Quantification of functionalised gold nanoparticle-targeted knockdown of gene expression in HeLa cells. In: PLoS ONE. 2014 ; Vol. 9, No. 6.
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Jiwaji, M, Sandison, ME, Reboud, J, Stevenson, R, Daly, R, Barkess, G, Faulds, K, Kolch, W, Graham, D, Girolami, MA, Cooper, JM & Pitt, A 2014, 'Quantification of functionalised gold nanoparticle-targeted knockdown of gene expression in HeLa cells' PLoS ONE, vol. 9, no. 6, e99458. https://doi.org/10.1371/journal.pone.0099458

Quantification of functionalised gold nanoparticle-targeted knockdown of gene expression in HeLa cells. / Jiwaji, Meesbah; Sandison, Mairi E.; Reboud, Julien; Stevenson, Ross; Daly, Rónán; Barkess, Gráinne; Faulds, Karen; Kolch, Walter; Graham, Duncan; Girolami, Mark A.; Cooper, Jonathan M.; Pitt, Andrew.

In: PLoS ONE, Vol. 9, No. 6, e99458, 13.06.2014.

Research output: Contribution to journalArticle

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T1 - Quantification of functionalised gold nanoparticle-targeted knockdown of gene expression in HeLa cells

AU - Jiwaji, Meesbah

AU - Sandison, Mairi E.

AU - Reboud, Julien

AU - Stevenson, Ross

AU - Daly, Rónán

AU - Barkess, Gráinne

AU - Faulds, Karen

AU - Kolch, Walter

AU - Graham, Duncan

AU - Girolami, Mark A.

AU - Cooper, Jonathan M.

AU - Pitt, Andrew

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PY - 2014/6/13

Y1 - 2014/6/13

N2 - Introduction: Gene therapy continues to grow as an important area of research, primarily because of its potential in the treatment of disease. One significant area where there is a need for better understanding is in improving the efficiency of oligonucleotide delivery to the cell and indeed, following delivery, the characterization of the effects on the cell. Methods: In this report, we compare different transfection reagents as delivery vehicles for gold nanoparticles functionalized with DNA oligonucleotides, and quantify their relative transfection efficiencies. The inhibitory properties of small interfering RNA (siRNA), single-stranded RNA (ssRNA) and single-stranded DNA (ssDNA) sequences targeted to human metallothionein hMT-IIa are also quantified in HeLa cells. Techniques used in this study include fluorescence and confocal microscopy, qPCR and Western analysis. Findings: We show that the use of transfection reagents does significantly increase nanoparticle transfection efficiencies. Furthermore, siRNA, ssRNA and ssDNA sequences all have comparable inhibitory properties to ssDNA sequences immobilized onto gold nanoparticles. We also show that functionalized gold nanoparticles can co-localize with autophagosomes and illustrate other factors that can affect data collection and interpretation when performing studies with functionalized nanoparticles. Conclusions: The desired outcome for biological knockdown studies is the efficient reduction of a specific target; which we demonstrate by using ssDNA inhibitory sequences targeted to human metallothionein IIa gene transcripts that result in the knockdown of both the mRNA transcript and the target protein.

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