Gold nanoparticle-based beacon to detect STAT5b mRNA expression in living cells: a case optimized by bioinformatics screen

Abstract

Messenger RNA (mRNA), a single-strand ribonucleic acid with functional gene information is usually abnormally expressed in cancer cells and has become a promising biomarker for the study of tumor progress. Hairpin DNA-coated gold nanoparticle (hDAuNP) beacon containing a bare gold nanoparticle (AuNP) as fluorescence quencher and thiol-terminated fluorescently labeled stem–loop–stem oligonucleotide sequences attached by Au–S bond is currently a new nanoscale biodiagnostic platform capable of mRNA detection, in which the design of the loop region sequence is crucial for hybridizing with the target mRNA. Hence, in this study, to improve the sensitivity and selectivity of hDAuNP beacon simultaneously, the loop region of hairpin DNA was screened by bioinformatics strategy. Here, signal transducer and activator of transcription 5b (STAT5b) mRNA was selected and used as a practical example. The results from the combined characterizations using optical techniques, flow cytometry assay, and cell microscopic imaging showed that after optimization, the as-prepared hDAuNP beacon had higher selectivity and sensitivity for the detection of STAT5b mRNA in living cells, as compared with our previous beacon. Thus, the bioinformatics method may be a promising new strategy for assisting in the designing of the hDAuNP beacon, extending its application in the detection of mRNA expression and the resultant mRNA-based biological processes and disease pathogenesis.

Keywords:

• molecular beacon
• bioinformatics
• gold nanoparticle
• STAT5b mRNA
• visual detection

Supplementary materials

Figure S1 TEM images of (A) initial AuNPs and (B) the resultant STAT5b hDAuNP beacon.

Note: These two TEM images demonstrate that after the formation of hDAuNP beacon, the average diameter of initial AuNPs was retained (15±1.3 nm).

Abbreviations: AuNP, gold nanoparticle; hDAuNP, hairpin DNA-coated gold nanoparticle; STAT5b, signal transducer and activator of transcription 5b; TEM, transmission electron microscopy.

Figure S2 Stability of STAT5b hDAuNP beacon.

Notes: Fluorescence spectra of STAT5b hDAuNP beacons before and after addition of DNase I or GSH, in which excess DTT was added to achieve the complete fluorescence recovery due to the thiol–thiol exchange reaction (this thiol–thiol exchange reaction will result in a complete release of thiol-terminated FITC-labeled hairpin DNA from AuNP surface). When DNase I or GSH was added, both the solutions of beacons exhibited low fluorescence signals.

Abbreviations: AuNP, gold nanoparticle; DNase, deoxyribonuclease; DTT, dithiothreitol; FITC, fluorescein isothiocyanate; GSH, glutathione; hDAuNP, hairpin DNA-coated gold nanoparticle; STAT5b, signal transducer and activators of transcription 5b.

Figure S3 Cytotoxicity of STAT5b hDAuNP beacon.

Notes: Cell viability was determined by the MTT assay following 24 hours of continuous exposure to various concentrations of hDAuNP beacon. STAT5b hDAuNP beacon did not show obvious cytotoxicity at concentrations up to 2.5 nM.

Abbreviations: hDAuNP, hairpin DNA-coated gold nanoparticle; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; STAT5b, signal transducer and activator of transcription 5b.

Figure S4 The vectors for RNA interference.

Notes: Upper panel: the scheme for the structure of the vector pGv113. Lower panel: The information for the shRNA sequence.

Abbreviations: shRNA, short hairpin RNA; STAT5B, signal transducer and activator of transcription 5b; PCMV, porcine cytomegalovirus; LTR, long terminal repeat; MCS, multiple clone site; RFP, red fluorescent protein; pBR ori, plasmid Bolivar Rodriguez origin; Ampr, ampicillin resistance.

Figure S5 Fluorescent images of the transfected MCF-7 cells.

Notes: Negative interference (CON055), transfection with empty vector pGv113; positive interference (22006), transfection with the pGv113-shRNA, in which the red fluorescence is from the transfected red fluorescent protein.

Abbreviation: shRNA, short hairpin RNA.

Figure S6 The experimental results obtained by previous STAT5b hDAuNP beacon 2.

Notes: (A) CLSM images of human STAT5b mRNA-expressing HepG-2 cells (upper panels) and nonhuman STAT5b-expressing PC12 cells (lower panels), treated with our previous STAT5b hDAuNP beacon 2. (B) The corresponding FC analysis of HepG-2 cells (mean fluorescence value: 4.5) and PC12 cells (mean fluorescence value: 2.7).

Abbreviations: CLSM, confocal laser scanning microscopy; FC, flow cytometry; hDAuNP, hairpin DNA-coated gold nanoparticle; STAT5b, signal transducer and activator of transcription 5b; FL1-H, height of fluorescence intensity; DIC, differential interference contrast microscopy.

Acknowledgments

This work was financially supported by the National Natural Science Foundation of China (grant numbers 81371627, 81220108012, 61335007, 81371684, 81171395, and 81328012).

Disclosure

The authors report no conflicts of interest in this work.