title: Visual detection of STAT5B gene expression in living cell using the hairpin DNA modified gold nanoparticle beacon.
abstract: Signal transducer and activator of transcription 5B (STAT5B) is an important protein in JAK-STAT signaling pathway that is responsible for the metastasis and proliferation of tumor cells. Determination of the STAT5B messenger Ribonucleic Acid (mRNA) relating to the STAT5B expression provides insight into the mechanism of tumor progression. In this study, we designed and used a special hairpin deoxyribonucleic acid (DNA) for human STAT5B mRNA to functionalize gold nanoparticles, which served as a beacon for detecting human STAT5B expression. Up to 90% quenching efficiency was achieved. Upon hybridizing with the target mRNA, the hairpin DNA modified gold nanoparticle beacons (hDAuNP beacons) release the fluorophores attached at 5' end of the oligonucleotide sequence. The fluorescence properties of the beacon before and after the hybridization with the complementary DNA were confirmed in vitro. The stability of hDAuNP beacons against degradation by DNase I and GSH indicated that the prepared beacon is stable inside cells. The detected fluorescence in MCF-7 cancer cells correlates with the specific STAT5B mRNA expression, which is consistent with the result from PCR measurement. Fluorescence microscopy showed that the hDAuNP beacons internalized in cells without using transfection agents, with intracellular distribution in the cytoplasm rather than the nucleus. The results demonstrated that this beacon could directly provide quantitative measurement of the intracellular STAT5B mRNA in living cells. Compared to the previous approaches, this beacon has advantages of higher target to background ratio of detection and an increased resistance to nuclease degradation. The strategy reported in this study is a promising approach for the intracellular measurement of RNA or protein expression in living cells, and has great potential in the study of drug screening and discovery.
authors.affiliation: Department of Biomedical Engineering, State Key Laboratory of Natural Medicines, School of Life Science and Technology, China Pharmaceutical University, Nanjing 210009, PR China.
authors.forename: Jianpeng
authors.initials: J
authors.lastname: Xue
authors.forename: Lingling
authors.initials: L
authors.lastname: Shan
authors.forename: Haiyan
authors.initials: H
authors.lastname: Chen
authors.forename: Yang
authors.initials: Y
authors.lastname: Li
authors.forename: Hongyan
authors.initials: H
authors.lastname: Zhu
authors.forename: Dawei
authors.initials: D
authors.lastname: Deng
authors.forename: Zhiyu
authors.initials: Z
authors.lastname: Qian
authors.forename: Samuel
authors.initials: S
authors.lastname: Achilefu
authors.forename: Yueqing
authors.initials: Y
authors.lastname: Gu
mesh_terms: D015374:Biosensing Techniques / Q000295:instrumentation*; D001943:Breast Neoplasms / Q000235:genetics / Q000378:metabolism*; D045744:Cell Line, Tumor; D004247:DNA / Q000737:chemistry* / Q000235:genetics*; D004867:Equipment Design; D019544:Equipment Failure Analysis; D020869:Gene Expression Profiling / Q000295:instrumentation*; D006801:Humans; D055029:Inverted Repeat Sequences / Q000235:genetics; D015336:Molecular Probe Techniques / Q000295:instrumentation*; D015335:Molecular Probes; D015203:Reproducibility of Results; D050799:STAT5 Transcription Factor / Q000235:genetics / Q000378:metabolism*; D012680:Sensitivity and Specificity
chemical_list: D015335:Molecular Probes; D050799:STAT5 Transcription Factor; C494095:STAT5B protein, human; D004247:DNA