Development of a novel method to detect intrinsic mRNA in a living cell by using a molecular beacon-immobilized nanoneedle.
We developed a novel nanobiosensor for monitoring mRNA expression in a single living cell by using an atomic force microscope (AFM) equipped with a nanoprobe. The nanoprobe was constructed by immobilizing a biotin-modified molecular beacon onto an ultrathin needle (nanoneedle) via neutravidin. In order to evaluate the effectiveness of the nanoprobe, we selected glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA as the target. A single HeLa cell contained approximately 1000 copies of GAPDH mRNA. The nanoprobe was directly inserted into living HeLa cells, and it reacted with the intrinsic target mRNA within the cells. The nanoprobe could be renatured by pulling it out of the cells. Further, we successfully used the nanoprobe for continuous detection of GAPDH mRNA in multiple cells, i.e., the nanoprobe was highly specific and sensitive for the detection of intrinsic mRNA in single living cells. mRNAs are thought to be highly condensed because of the large number of organelles and complexes present in cells and the limited space available for distribution. Therefore, direct analysis of intrinsic mRNAs in living cells would be advantageous, and our novel nanoprobe is highly suitable for monitoring the RNAs in living cells.
Kihara
Takanori
T
Department of Bioengineering, School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan.
Yoshida
Narutoshi
N
Kitagawa
Taro
T
Nakamura
Chikashi
C
Nakamura
Noriyuki
N
Miyake
Jun
J
D001483:Base Sequence; D015374:Biosensing Techniques / Q000295:instrumentation / Q000379:methods*; D001710:Biotin; D015342:DNA Probes / Q000235:genetics; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D049329:Nanostructures; D012333:RNA, Messenger / Q000032:analysis* / Q000235:genetics*; D012825:Silicon; D059010:Single-Cell Analysis / Q000295:instrumentation / Q000379:methods*
D015342:DNA Probes; D012333:RNA, Messenger; D001710:Biotin; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D012825:Silicon