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DNA-based sensor for real-time measurement of the enzymatic activity of human topoisomerase I.

Marcussen LB, Jepsen ML, Kristoffersen EL, Franch O, Proszek J, Ho YP, Stougaard M, Knudsen BR - Sensors (Basel) (2013)

Bottom Line: The cytotoxic effect of camptothecins correlates directly with the intracellular topoisomerase I activity.We therefore envision that the presented sensor may find use for the prediction of cellular drug response.Moreover, inhibition of topoisomerase I by camptothecin is readily detectable using the presented DNA sensor, suggesting a potential application of the sensor for first line screening for potential topoisomerase I targeting anti-cancer drugs.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology and Genetics, Aarhus University, Aarhus C 8000, Denmark. laerke_bay@hotmail.com

ABSTRACT
Sensors capable of quantitative real-time measurements may present the easiest and most accurate way to study enzyme activities. Here we present a novel DNA-based sensor for specific and quantitative real-time measurement of the enzymatic activity of the essential human enzyme, topoisomerase I. The basic design of the sensor relies on two DNA strands that hybridize to form a hairpin structure with a fluorophore-quencher pair. The quencher moiety is released from the sensor upon reaction with human topoisomerase I thus enabling real-time optical measurement of enzymatic activity. The sensor is specific for topoisomerase I even in raw cell extracts and presents a simple mean of following enzyme kinetics using standard laboratory equipment such as a qPCR machine or fluorimeter. Human topoisomerase I is a well-known target for the clinically used anti-cancer drugs of the camptothecin family. The cytotoxic effect of camptothecins correlates directly with the intracellular topoisomerase I activity. We therefore envision that the presented sensor may find use for the prediction of cellular drug response. Moreover, inhibition of topoisomerase I by camptothecin is readily detectable using the presented DNA sensor, suggesting a potential application of the sensor for first line screening for potential topoisomerase I targeting anti-cancer drugs.

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(A) Shows a bar chart depicting the initial velocity calculated from of three individual experiments where the DNA sensor were incubated with crude cell extracts from yeast S. cerevisiae expressing (Yeast extr+TopI) or not expressing (Yeast extr–TopI) hTopI. (B) Bar chart depicting the initial velocity of reactions performed in the absence or presence of 50 or 75 μM CPT. In both parts of the figure, data were normalized against the maximum initial velocity (corresponding to 100%) obtained in each repetition
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f3-sensors-13-04017: (A) Shows a bar chart depicting the initial velocity calculated from of three individual experiments where the DNA sensor were incubated with crude cell extracts from yeast S. cerevisiae expressing (Yeast extr+TopI) or not expressing (Yeast extr–TopI) hTopI. (B) Bar chart depicting the initial velocity of reactions performed in the absence or presence of 50 or 75 μM CPT. In both parts of the figure, data were normalized against the maximum initial velocity (corresponding to 100%) obtained in each repetition

Mentions: In order to address the potential use of the DNA sensor for such purposes we investigated the specificity of the sensor towards hTopI in crude biological samples. As a model system we used whole cell extracts from the yeast strain RS190 (lacking the endogenous TOP1 gene) with or without an episomal plasmid that supports the expression of recombinant hTopI. Following preparation of raw cell extracts, with or without hTopI activity, the extracts were incubated with the sensor and the reaction measured in terms of fluorescence emission in a qPCR machine. The initial velocity of the reaction observed in the two cell extracts were calculated as described for Figure 2 and the mean of the results obtained from three individual experiments depicted in Figure 3(A). The complete lack of activity observed in extract without hTopI strongly supports the specificity of the DNA sensor towards hTopI activity in raw cell extracts under the utilized reaction conditions.


DNA-based sensor for real-time measurement of the enzymatic activity of human topoisomerase I.

Marcussen LB, Jepsen ML, Kristoffersen EL, Franch O, Proszek J, Ho YP, Stougaard M, Knudsen BR - Sensors (Basel) (2013)

(A) Shows a bar chart depicting the initial velocity calculated from of three individual experiments where the DNA sensor were incubated with crude cell extracts from yeast S. cerevisiae expressing (Yeast extr+TopI) or not expressing (Yeast extr–TopI) hTopI. (B) Bar chart depicting the initial velocity of reactions performed in the absence or presence of 50 or 75 μM CPT. In both parts of the figure, data were normalized against the maximum initial velocity (corresponding to 100%) obtained in each repetition
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3673067&req=5

f3-sensors-13-04017: (A) Shows a bar chart depicting the initial velocity calculated from of three individual experiments where the DNA sensor were incubated with crude cell extracts from yeast S. cerevisiae expressing (Yeast extr+TopI) or not expressing (Yeast extr–TopI) hTopI. (B) Bar chart depicting the initial velocity of reactions performed in the absence or presence of 50 or 75 μM CPT. In both parts of the figure, data were normalized against the maximum initial velocity (corresponding to 100%) obtained in each repetition
Mentions: In order to address the potential use of the DNA sensor for such purposes we investigated the specificity of the sensor towards hTopI in crude biological samples. As a model system we used whole cell extracts from the yeast strain RS190 (lacking the endogenous TOP1 gene) with or without an episomal plasmid that supports the expression of recombinant hTopI. Following preparation of raw cell extracts, with or without hTopI activity, the extracts were incubated with the sensor and the reaction measured in terms of fluorescence emission in a qPCR machine. The initial velocity of the reaction observed in the two cell extracts were calculated as described for Figure 2 and the mean of the results obtained from three individual experiments depicted in Figure 3(A). The complete lack of activity observed in extract without hTopI strongly supports the specificity of the DNA sensor towards hTopI activity in raw cell extracts under the utilized reaction conditions.

Bottom Line: The cytotoxic effect of camptothecins correlates directly with the intracellular topoisomerase I activity.We therefore envision that the presented sensor may find use for the prediction of cellular drug response.Moreover, inhibition of topoisomerase I by camptothecin is readily detectable using the presented DNA sensor, suggesting a potential application of the sensor for first line screening for potential topoisomerase I targeting anti-cancer drugs.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology and Genetics, Aarhus University, Aarhus C 8000, Denmark. laerke_bay@hotmail.com

ABSTRACT
Sensors capable of quantitative real-time measurements may present the easiest and most accurate way to study enzyme activities. Here we present a novel DNA-based sensor for specific and quantitative real-time measurement of the enzymatic activity of the essential human enzyme, topoisomerase I. The basic design of the sensor relies on two DNA strands that hybridize to form a hairpin structure with a fluorophore-quencher pair. The quencher moiety is released from the sensor upon reaction with human topoisomerase I thus enabling real-time optical measurement of enzymatic activity. The sensor is specific for topoisomerase I even in raw cell extracts and presents a simple mean of following enzyme kinetics using standard laboratory equipment such as a qPCR machine or fluorimeter. Human topoisomerase I is a well-known target for the clinically used anti-cancer drugs of the camptothecin family. The cytotoxic effect of camptothecins correlates directly with the intracellular topoisomerase I activity. We therefore envision that the presented sensor may find use for the prediction of cellular drug response. Moreover, inhibition of topoisomerase I by camptothecin is readily detectable using the presented DNA sensor, suggesting a potential application of the sensor for first line screening for potential topoisomerase I targeting anti-cancer drugs.

Show MeSH
Related in: MedlinePlus