Interactions of Potential Protein Cancer Biomarker Survivin with Plasmonic Nanoparticles and Its Dynamics in Cancer Cell
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Interactions of Potential Protein Cancer Biomarker Survivin with Plasmonic Nanoparticles and Its Dynamics in Cancer Cells Studied Using Fluorescence Molecular-Beacon Probes, Gated-RET and EQCN Methods Magdalena Stobiecka*1, Agata Chalupa2, Beata Dworakowska1 1
Department of Biophysics, Warsaw University of Life Sciences (SGGW), Warsaw 02776,
Poland. 2
Institute of Nanoparticle Nanocarriers, Barczewo 11010, Poland.
ABSTRACT The protein survivin (Sur) has been considered as a potential cancer biomarker due to its involvement in disrupting normal cell cycle by stimulating proliferation and inhibiting cell apoptosis. In this work, we have focused on exploring novel platforms for sensitive monitoring of Sur expression, based on molecular beacons and protein modulation of plasmon-controlled fluorescence. In this framework, we show that Sur can be employed in gating the resonance energy transfer (gRET) between fluorescein isothiocyanate probe dye (FITC) and plasmonic citrate-capped gold nanoparticles (AuNP@Cit). Furthermore, we have designed fluorescent dyebearing molecular beacons (MBs) targeting nucleotides of the survivin mRNA. The antisense oligonucleotide complementary to the target sequence, inserted in the loop area of the hairpin MB structure, has enabled the fluorescence turn-ON MB switching in the presence of the target, thus signaling the high Sur mRNA levels and enhanced Sur protein expression. INTRODUCTION Survivin (Sur) is the smallest member of the inhibitor of apoptosis proteins family (IAP). It plays a crucial role in the regulation of cell division, cell cycle control, and in the inhibition of apoptosis. Sur is intensely expressed in the embryonic tissue, fetal organs, in a variety of human malignancies and tumors but is almost undetectable in the normal differentiated cells [1-4]. Survivin protein and survivin mRNA have recently been extensively studied in view of their utilization as selective targets for cancer therapy and are considered as significant tumor markers [2, 4, 5]. Methods used for Sur detection are mostly based on molecular biology techniques including immunohistochemical methods [6], Western blot (WB), ELISA analysis [7], and RTPCR [8]. Most recently, survivin mRNA in living cells was also analyzed using optical, fluorescence and electrochemical biosensors [9-11]. Liu et al. [10] have designed an electrochemical sensor based on dually labeled stem-loop DNA structure attached to a gold electrode to monitor the survivin mRNA in human hepatoma cell line (SMMC-7721). Li et al
[11] used antibody-based microfluidic devices. They estimated the survivin mRNA content in single captured prostate cancer cell as (4.8±1.8)×106 copies. In this work, we have focused on designing and testing fluorescent molecular beacons (MBs) targeting the BIRC5 gene which is responsible for the expression of Sur. The antisense oligonucleotides complementary to the target sequence of Sur mRNA have been inserted in the loop area of a hairpin MB structure. To evaluate the interactions of the Sur protein with plasmon
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