Rolling circle amplification-based detection of human topoisomerase I activity on magnetic beads

2014 ◽  
Vol 451 ◽  
pp. 42-44 ◽  
Author(s):  
Laura Zuccaro ◽  
Cinzia Tesauro ◽  
Barbara Cerroni ◽  
Alessio Ottaviani ◽  
Birgitta Ruth Knudsen ◽  
...  
Keyword(s):  
Topoisomerase I ◽  
Magnetic Beads ◽  
Rolling Circle Amplification ◽  
Rolling Circle

Molecular Beacon Enables Combination of Highly Processive and Highly Sensitive Rolling Circle Amplification Readouts for Detection of DNA-Modifying Enzymes

Nano LIFE ◽  
2015 ◽  
Vol 05 (02) ◽  
pp. 1541002 ◽  
Author(s):  
Emil L. Kristoffersen ◽  
Maria Gonzalez ◽  
Magnus Stougaard ◽  
Cinzia Tesauro
Keyword(s):  
Real Time ◽  
Dna Cleavage ◽  
Topoisomerase I ◽  
Drug Response ◽  
Molecular Beacon ◽  
Rolling Circle Amplification ◽  
Dna Topoisomerase ◽  
Rolling Circle ◽  
Topoisomerase Ib ◽  
Highly Sensitive

Here we present an optimized readout format for detection of the circularized products from a DNA-based sensor for measurement of DNA-modifying enzymes including DNA Topoisomerase I. The basic design of the DNA-sensor relies on the use of a substrate that can be circularized by the activity of DNA-modifying enzymes like type IB Topoisomerases and subsequently amplified by a rolling circle amplification (RCA) mechanism. The RCA process can be followed in real-time by the addition of a molecular beacon with a fluorophore/quencher pair. Upon hybridization to the amplified product, the fluorophore/quencher pair is separated, giving rise to a fluorescent signal, measurable in pseudo real-time using a qPCR machine or in a fluorimeter. The RCA products in complex with the molecular beacon can subsequently be moved to microscopic slides and analyzed in a fluorescence microscope. We describe the proof of the principle of this molecular beacon-based method combining the qPCR readout format with the standard Rolling circle Enhanced Enzymatic Assay previously reported. Although the qPCR setup is less sensitive, it allows easy, fast, and high-throughput measurement of enzyme activities. Human Topoisomerase IB (TopIB) is a well-known target for the clinically used anticancer drugs of the camptothecin family. The cytotoxic effect of camptothecins correlates directly with the intracellular TopIB activity affecting reversibly the Topoisomerase/DNA cleavage complexes. Therefore, we envisioned that the presented method may find use for the prediction of cellular drug response and for drug screening to discover novel molecules that specifically inhibit TopIB or other DNA-modifying enzymes.

Cocaine detection via rolling circle amplification of short DNA strand separated by magnetic beads

2011 ◽  
Vol 26 (7) ◽  
pp. 3309-3312 ◽  
Author(s):  
Cuiping Ma ◽  
Wenshuo Wang ◽  
Qing Yang ◽  
Chao Shi ◽  
Lijie Cao
Keyword(s):  
Magnetic Beads ◽  
Rolling Circle Amplification ◽  
Rolling Circle ◽  
Dna Strand ◽  
Cocaine Detection

Liposome-encoded magnetic beads initiated by padlock exponential rolling circle amplification for portable and accurate quantification of microRNAs

2017 ◽  
Vol 53 (78) ◽  
pp. 10772-10775 ◽  
Author(s):  
Qingwang Xue ◽  
Yancong Kong ◽  
Huaisheng Wang ◽  
Wei Jiang
Keyword(s):  
Magnetic Beads ◽  
Rolling Circle Amplification ◽  
Rolling Circle ◽  
Accurate Quantification

In this work, we propose a strategy for glucoamylase-encapsulated liposome-encoded magnetic beads initiated by padlock exponential rolling circle amplification (P-ERCA) for portable and accurate quantification of miRNA by using a glucometer (GM) for readout.

scholarly journals On the Electrochemical Detection of Alpha-Fetoprotein Using Aptamers: DNA Isothermal Amplification Strategies to Improve the Performance of Weak Aptamers

Biosensors ◽  
2020 ◽  
Vol 10 (5) ◽  
pp. 46
Author(s):  
Ramón Lorenzo-Gómez ◽  
Daniel González-Robles ◽  
Rebeca Miranda-Castro ◽  
Noemí de-los-Santos-Álvarez ◽  
María Jesús Lobo-Castañón
Keyword(s):  
Dissociation Constant ◽  
Electrochemical Detection ◽  
Magnetic Beads ◽  
Rolling Circle Amplification ◽  
Binding Constants ◽  
Alpha Fetoprotein ◽  
Isothermal Amplification ◽  
Terminal Deoxynucleotidyl Transferase ◽  
Tumor Biomarker ◽  
Rolling Circle

Affinity characterization is essential to develop reliable aptamers for tumor biomarker detection. For alpha-fetoprotein (AFP), a biomarker of hepatocellular carcinoma (HCC), two DNA aptamers were described with very different affinity. In this work, we estimate the dissociation constant of both of them by means of a direct assay on magnetic beads modified with AFP and electrochemical detection on carbon screen-printed electrodes (SPCE). Unlike previous works, both aptamers showed similar dissociation constant (Kd) values, in the subµM range. In order to improve the performance of these aptamers, we proposed the isothermal amplification of the aptamers by both terminal deoxynucleotidyl transferase (TdT) and rolling circle amplification (RCA). Both DNA amplifications improved the sensitivity and also the apparent binding constants from 713 nM to 189 nM for the short aptamer and from 526 nM to 32 nM for the long aptamer. This improvement depends on the true affinity of the binding pair, which ultimately limits the analytical usefulness.

scholarly journals Improving the Analytical Performance of Weak Aptamers: DNA Isothermal Amplification Approaches

Proceedings ◽  
2020 ◽  
Vol 60 (1) ◽  
pp. 26
Author(s):  
Ramón Lorenzo-Gómez ◽  
Rebeca Miranda-Castro ◽  
Noemí de-los-Santos-Álvarez ◽  
María Jesús Lobo-Castañón
Keyword(s):  
Magnetic Beads ◽  
Rolling Circle Amplification ◽  
Binding Constants ◽  
Isothermal Amplification ◽  
Terminal Deoxynucleotidyl Transferase ◽  
Tumor Biomarker ◽  
Dna Aptamers ◽  
Rolling Circle ◽  
Neutrophil Gelatinase Associated Lipocalin ◽  
Potential Biomarker

Affinity characterization is an essential but time-consuming task to develop reliable aptamers for tumor biomarker detection and is not always thoroughly addressed. For neutrophil gelatinase-associated lipocalin (NGAL), a potential biomarker of pancreatic cancer, two DNA aptamers were described with very different affinity. Likewise, another pair of DNA aptamers was developed with very different affinity for alpha-fetoprotein (AFP), a biomarker of hepatocellular carcinoma. In this work, we estimated the dissociation constant of these aptamers by means of a direct assay on magnetic beads modified with biomarker and electrochemical detection on screen-printed carbon electrodes. In order to improve the performance of these aptamers, we proposed the isothermal amplification of the aptamers for both biomarkers by rolling circle amplification (RCA). In the case of AFP aptamers, we also tried terminal deoxynucleotidyl transferase (TdT), a template-independent amplification. Both DNA amplifications improved the sensitivity and the apparent binding constants of the aptamers for the two cancer biomarkers. Nevertheless, this improvement depends on the true affinity of the binding pair, which ultimately limits their analytical usefulness.

Sign in / Sign up

Export Citation Format

Share Document