Quantum Dot-Conjugated Hybridization Probes for Preliminary Screening of siRNA Sequences

• Published in: Journal of the American Chemical Society Vol. 127; no. 32; pp. 11328 - 11335
• Main Authors: Bakalova, Rumiana, Zhelev, Zhivko, Ohba, Hideki, Baba, Yoshinobu
• Format: Journal Article
• Language: English
• Published: Washington, DC American Chemical Society 17.08.2005
• Subjects: Base Sequence; Biological and medical sciences; Carbocyanines - analysis; Carbocyanines - chemistry; Fluorescence Resonance Energy Transfer; Fundamental and applied biological sciences. Psychology; Gene expression; Molecular and cellular biology; Molecular genetics; Nucleic Acid Hybridization - methods; Quantum Dots; RNA Probes - chemistry; RNA, Messenger - chemistry; RNA, Small Interfering - chemistry; Sequence Analysis, RNA; Molecular hybridization; Gene silencing; Sequence specificity; Semiconductor quantum dots; Interference; Resonance energy; Oligonucleotide; Single stranded RNA; Energy transfer; Fluorescence spectrometry
• ISSN: 0002-7863; 1520-5126
• DOI: 10.1021/ja051089h

In the present study, we describe the design and fabrication of quantum dot-conjugated hybridization probes and their application to the development of a comparatively simple and rapid procedure for the selection of highly effective small-interfering RNA (siRNA) sequences for RNA interference (RNAi) in mammalian cells, for example, siRNAs with high accessibility and affinity to the respective mRNA target. A single-stranded siRNA was conjugated with a quantum dot and used as a hybridization probe. The target mRNA was amplified in the presence of Cy5-labeled nucleotides, and Cy5−mRNA served as a hybridization sample. The formation of siRNA/mRNA duplexes during a comparatively short hybridization time (1 h) was used as a criterion for the selection of highly effective, target-specific siRNA sequences. The accessibility and affinity of the siRNA sequence for the target mRNA site were determined by fluorescence resonance energy transfer (FRET) between a quantum dot (donor) and a fluorescent dye molecule (Cy5, acceptor) localized at an appropriate distance from each other when hybridization occurred. The FRET signal was observed only when there was high accessibility between an antisense siRNA and a sense mRNA and did not appear in the case of mismatch siRNAs. Moreover, the amplitude of the FRET signal significantly correlated with the specific effect of siRNA on the expression of the target mRNA and protein, determined in native cells by RT-PCR and immunoblot analysis, respectively.