TY - JOUR
T1 - Gold-nanobeacons for real-time monitoring of RNA synthesis
AU - Rosa, João
AU - Conde, João
AU - de la Fuente, Jesus M.
AU - Lima, João C.
AU - Baptista, Pedro V.
N1 - This work was supported by Fundacao para a Ciencia e Tecnologia [CIGMH, REQUIMTE, PTDC/QUI-QUI/112597/2009, SFRH/BD/43320/2008 to J.R., SFRH/BD/62957/2009 to J.C.]; Nanotruck-Action from NanoSciEra+; ARAID to J.M.F.
PY - 2012/6/1
Y1 - 2012/6/1
N2 - Measuring RNA synthesis and, when required, the level of inhibition, is crucial towards the development of practical strategies to evaluate silencing efficiency of gene silencing approaches. We developed a direct method to follow RNA synthesis in real time based on gold nanoparticles (AuNPs) functionalized with a fluorophore labeled hairpin-DNA, i.e. gold-nanobeacon (Au-nanobeacon). Under hairpin configuration, proximity to gold nanoparticles leads to fluorescence quenching; hybridization to a complementary target restores fluorescence emission due to the Au-nanobeacons' conformational reorganization that causes the fluorophore and the AuNP to part from each other, yielding a quantitative response. With this reporter Au-nanobeacon we were able to measure the rate of in vitro RNA synthesis (~10.3. fmol of RNA per minute). Then, we designed a second Au-nanobeacon targeting the promoter sequence (inhibitor) so as to inhibit transcription whilst simultaneously monitor the number of promoters being silenced. Using the two Au-nanobeacons in the same reaction mixture, we are capable of quantitatively assess in real time the synthesis of RNA and the level of inhibition.The biosensor concept can easily be extended and adapted to situations when real-time quantitative assessment of RNA synthesis and determination of the level of inhibition are required. In fact, this biosensor may assist the in vitro evaluation of silencing potential of a given sequence to be later used for in vivo gene silencing.
AB - Measuring RNA synthesis and, when required, the level of inhibition, is crucial towards the development of practical strategies to evaluate silencing efficiency of gene silencing approaches. We developed a direct method to follow RNA synthesis in real time based on gold nanoparticles (AuNPs) functionalized with a fluorophore labeled hairpin-DNA, i.e. gold-nanobeacon (Au-nanobeacon). Under hairpin configuration, proximity to gold nanoparticles leads to fluorescence quenching; hybridization to a complementary target restores fluorescence emission due to the Au-nanobeacons' conformational reorganization that causes the fluorophore and the AuNP to part from each other, yielding a quantitative response. With this reporter Au-nanobeacon we were able to measure the rate of in vitro RNA synthesis (~10.3. fmol of RNA per minute). Then, we designed a second Au-nanobeacon targeting the promoter sequence (inhibitor) so as to inhibit transcription whilst simultaneously monitor the number of promoters being silenced. Using the two Au-nanobeacons in the same reaction mixture, we are capable of quantitatively assess in real time the synthesis of RNA and the level of inhibition.The biosensor concept can easily be extended and adapted to situations when real-time quantitative assessment of RNA synthesis and determination of the level of inhibition are required. In fact, this biosensor may assist the in vitro evaluation of silencing potential of a given sequence to be later used for in vivo gene silencing.
KW - Fluorophore labeled hairpin-DNA
KW - Gold nanoparticles
KW - Gold-nanobecons
KW - Inhibition of transcription
KW - RNA synthesis
UR - http://www.scopus.com/inward/record.url?scp=84861653867&partnerID=8YFLogxK
U2 - 10.1016/j.bios.2012.04.006
DO - 10.1016/j.bios.2012.04.006
M3 - Article
C2 - 22541812
AN - SCOPUS:84861653867
VL - 36
SP - 161
EP - 167
JO - Biosensors & Bioelectronics
JF - Biosensors & Bioelectronics
SN - 0956-5663
IS - 1
ER -