ABSTRACT
Telomerase has been regarded as a biomarker for cancer diagnosis as well as the clinical treatment and the reliable detection of intracellular telomerase activity is of great significance. By developing a telomere elongation-based DNA-catalytic amplification strategy, a novel surface-enhanced Raman scattering (SERS) method is proposed for the assay of telomerase activity. In the presence of telomerase and nucleotide mixture dNTPs, the telomerase substrate (TS) primer extended and generated a long single-strand DNA (ssDNA) containing the telomere repeat units (TTAGGG)n, which could catalyze the entropy-driven circuit reaction (EDCR). One of the products of EDCR was ingeniously used as the catalyst of catalytic hairpin assembly (CHA) occured on magnetic beads (MBs). As a result, a large amount of ROX-labeled Raman probes could be anchored on the surface of MBs and used for SERS detection. Using this strategy, the assay can detect telomerase activity from cell extracts equivalent down to single HeLa cell.
Subject(s)
Biosensing Techniques/methods , DNA, Catalytic/metabolism , Spectrum Analysis, Raman/methods , Telomerase/metabolism , Cell Line, Tumor , Drug Evaluation, Preclinical , Enzyme Assays/methods , Humans , Nucleic Acid Amplification Techniques/methods , Substrate Specificity , Telomerase/antagonists & inhibitors , Telomere/metabolismABSTRACT
Nucleic acid detection with label-free biosensors circumvents costly fluorophore functionalization steps associated with conventional assays by utilizing transducers of impressive ultimate detection limits. Despite this technological prowess, molecular recognition at a surface limits the biosensors' sensitivity, specificity, and reusability. It is therefore imperative to integrate novel molecular approaches with existing label-free transducers to overcome those limitations. Here, we demonstrate this concept by integrating a DNA strand displacement circuit with a micron-scale whispering gallery mode (WGM) microsphere biosensor. The integrated biosensor exhibits at least 25-fold improved nucleic acid sensitivity, and sets a new record for label-free microcavity biosensors by detecting 80 pM (32 fmol) of a 22nt oligomer; this improvement results from the catalytic behavior of the circuit. Furthermore, the integrated sensor exhibits extremely high specificity; single nucleotide variants yield 40- to 100-fold lower signal. Finally, the same physical sensor was demonstrated to alternatingly detect 2 different nucleic acid sequences through 5 cycles of detection, showcasing both its reusability and its versatility.
Subject(s)
Biosensing Techniques/instrumentation , DNA/analysis , DNA/genetics , Oligonucleotide Array Sequence Analysis/instrumentation , Sequence Analysis, DNA/instrumentation , Surface Plasmon Resonance/instrumentation , Base Sequence , Catalysis , DNA/chemistry , Equipment Design , Equipment Failure Analysis , Molecular Sequence Data , Reproducibility of Results , Sensitivity and Specificity , Staining and Labeling , Systems IntegrationABSTRACT
Objective To observe the suppression of HBsAg and HBeAg expression by DNAzyme and LNAzyme located at HBV pre-area of HBV.Methods Eecoding sequence of(10-23 DNAzyme) thiolmodificated 10-23DNAzyme and LNAzyme that were directed against Pre C/C region of HBV were designed and synthesized.Experimental groups and control groups were set up.The experimental groups included 10-23 DNAzyme group,(S-10-23) DNAzyme group and LNAzyme group.The control groups include blank control group,simple lipofectamine group,simple 10-23DNAzyme group and random 10-23 DNAzyme group.In the dosege of 0.16,0.64,1.28,1.60,(1.92 ?mol?L~(-1)) and the time of 12,24,36,48,60,72,84 and 96 h,the suppression of HBsAg and HBeAg expression by 10-23 DNAzyme and LNAzyme in 2.2.15 cells were studied.Results The suppression of HBsAg and HBeAg expression by 10-23 DNAzyme and LNAzyme in 2.2.15 cells were significant.The inhibitory effects caused by LNAzyme was more significant than that by thiolmodified 10-23 DNAzyme whose inhibitory effects were more significant than that of 10-23 DNAzyme.The inhibitory rates of LNAzyme and 10-23 DNAzyme thiolmodification reached(91.6?8.4)%,(78.4?2.0)% on HBsAg,respectivelly and(90.1?5.2)%,(76.4?4.8)% on HBeAg.The inhibitory effects of LNAzyme and thiolmodification of 10-23 DNAzyme were found 12 h after they were added to 2.2.15 cells,and optimized at 48 h,effective inhibitory time for LNAzyme was 84 h,for thiolmodification 10-23 DNAzyme was 72 h.Addition of LNAzyme and 10-23 DNAzyme to 2.2.15 cells didn′t exert cytotoxicity.Conclusion 10-23 DNAzyme and LNAzyme have demonstrated significant inhibitory effects on the HBsAg and HBeAg expressions in 2.2.15 cells.Morever,the inhibitory effects of LNAzyme is more significant than that of DNAzyme.LNAzyme is a specific anti-HBV therapeutic agent.
ABSTRACT
AIM: To study the inhibitory effects of 10-23 deoxyribozyme (10-23 DRz) on Escherichia coli ?-lactamase gene expression. METHODS: According to the gene sequence of Escherichia coli ?-lactamase gene blashv-5, 10-23 DRz and antisense oligonucleotides (As-ODN) were designed and synthesized. 10-23 DRz, As-ODN or control oligonucleotides were respectively introduced into Escherichia coli by the method of electroporation. Following electroporation, bacterial viability in liquid medium contained ceftazidime was detected, bacterial ?-lactamase expression was analysed by using IEF-PAGE and the ?-lactamase band was measured with gel documentation-analyzing system. RESULTS: A_ 600 in 10-23 DRz transfected Escherichia coli was lower compared with that in As-ODN transfected Escherichia coli (P
