Hairpin–Hairpin Molecular Beacon Interactions for Detection of Survivin mRNA in Malignant SW480 Cells

Abstract

Cancer biomarkers offer unique prospects for the development of cancer diagnostics and therapy. One of such biomarkers, protein survivin (Sur), exhibits strong antiapoptotic and proliferation-enhancing properties and is heavily expressed in multiple cancers. Thus, it can be utilized to provide new modalities for modulating the cell-growth rate, essential for effective cancer treatment. Herein, we have focused on the development of a new survivin-based cancer detection platform for colorectal cancer cells SW480 using a turn-on fluorescence oligonucleotide molecular beacon (MB) probe, encoded to recognize Sur messenger RNA (mRNA). Contrary to the expectations, we have found that both the complementary target oligonucleotide strands as well as the single- and double-mismatch targets, instead of exhibiting the anticipated simple random conformations, preferentially formed secondary structure motifs by folding into small-loop hairpin structures. Such a conformation may interfere with, or even undermine, the biorecognition process. To gain better understanding of the interactions involved, we have replaced the classical Tyagi-Kramer model of interactions between a straight target oligonucleotide strand and a hairpin MB with a new model to account for the hairpin-hairpin interactions as the biorecognition principle. A detailed mechanism of these interactions has been proposed. Furthermore, in experimental work, we have demonstrated an efficient transfection of malignant SW480 cells with SurMB probes containing a fluorophore Joe (SurMB-Joe) using liposomal nanocarriers. The green emission from SurMB-Joe in transfected cancer cells, due to the hybridization of the SurMB-Joe loop with Sur mRNA hairpin target, corroborates Sur overexpression. On the other hand, healthy human-colon epithelial cells CCD 841 CoN show only negligible expression of survivin mRNA. These experiments provide the proof-of-concept for distinguishing between the cancer and normal cells by the proposed hairpin-hairpin interaction method. The single nucleotide polymorphism sensitivity and a low detection limit of 26 nM (S/N = 3σ) for complementary targets have been achieved.

Optical Biosensing System for the Detection of Survivin mRNA in Colorectal Cancer Cells Using a Graphene Oxide Carrier-Bound Oligonucleotide Molecular Beacon

“…The inner filter effect observed at higher concentrations of GO is due to the high absorption of UV–Vis light by the GO nanocarriers, leading to the decrease of the intensity of the excitation beam in measurements of SurMB fluorescence in comparison to the case of the absence of GO. Results of the experiments described above validate the GO nanocarriers ability to transfer an oligonucleotide payload through a cell membrane, as well as corroborate the hairpin–hairpin interaction model [ 8 ] for GO-bound MBs in desorptive hybridization with hairpin mRNA targets, which is an extension of the original Tyagi–Kramer model developed for MBs interacting with linear oligonucleotide targets.…”

“…The ability of a hairpin MB to interact with a hairpin-structured target oligonucleotide (tDNA) has recently been demonstrated [ 8 ], and a hairpin–hairpin interaction model was proposed to extend the Tyagi–Kramer model [ 22 ] of molecular beacon interactions with a linear target oligonucleotide. Our calculations—performed using the UNAFold software—indicate that the target oligonucleotide (tDNA), complementary to the loop of SurMB, preferentially forms a stable small-loop hairpin structure instead of relaxing to a linear conformation.…”

“…From among the various nanocarriers studied for delivery systems, including gold nanoparticles [ 4 , 5 , 6 ], liposomes [ 7 , 8 , 9 , 10 ], micelles [ 11 ], exosomes [ 12 ], graphene oxide [ 2 , 13 , 14 , 15 , 16 , 17 , 18 , 19 ] and others [ 20 , 21 ], herein we focus specifically on the binding of molecular beacon (MB) probes to graphene oxide (GO) nanosheet nanocarriers and the mechanism of hybridization of the GO-bound molecular beacon (GO-MB) with target strands. Recently, we have demonstrated the ability of a hairpin MB to interact with a hairpin-structured target oligonucleotide [ 8 ], thus extending the Tyagi–Kramer model [ 22 ] of MB interaction with linear oligonucleotides. Therefore, the mechanism considered here is the hairpin–hairpin interaction of GO-bound MB with target strands, followed by the duplex DNA desorption from GO nanocarriers [ 23 , 24 , 25 , 26 ].…”

Monitoring of dynamic ATP level changes by oligomycin-modulated ATP synthase inhibition in SW480 cancer cells using fluorescent “On-Off” switching DNA aptamer

“…In this scheme, a transfection of SW480 cells with Apt(ATP) using Lipofectamine carriers, Apt(ATP)@Lip, is shown. Thus, the ATP aptamer is delivered via a liposomal endocytosis to SW480 cells [60]. The Apt(ATP) with FAM fluorescence tag generates a strong green emission signal induced by the presence of intracellular ATP.…”

MHY440, a Novel Topoisomerase Ι Inhibitor, Induces Cell Cycle Arrest and Apoptosis via a ROS-Dependent DNA Damage Signaling Pathway in AGS Human Gastric Cancer Cells

“…The excessive production of ROS in the mitochondria is known to play an important role in the regulation of apoptosis [33]. The downregulation of survivin, a member of inhibitor of apoptosis and an antagonist of apoptosis, was associated with ROS production in cancer cell apoptosis [34,35,36]. Some anticancer agents, such as cisplatin, doxorubicin, mitomycin C, and etoposide, are at least partially effective through the induction of ROS [37].…”