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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) Schematic illustration of the DNA sensor. The sensor is composed of a Cl strand that folds into a hairpin structure and contains an internal 6-FAM “F” and a 3′-BHQ1 “Q” moiety. The Cl is hybridized to a L strand with a 5′-OH end. The cleavage site is indicated by an arrow. (B) Flow chart of the anticipated reaction of hTopI with the DNA sensor. Cleavage results in release of the BHQ1 shifting the sensor to the ON state. Subsequently, ligation of the L strand releases hTopI and leaves the enzyme ready for another round of catalysis. The position of the fluorephore-quencher pair resulted in a quenching efficiency of approximately 80% (data not shown). (C) Gel picture showing the products resulting from incubating the DNA sensor with hTopI followed by trypsin- (lane 2) or proteinase K digestion (lane 3) or no protease treatment (lane 4). Lane 1 shows the gel-electrophoretic mobility of the unreacted sensor. The lengths (in bases) of the substrate and the upper product are indicted to the left of the gel-picture.
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f1-sensors-13-04017: (A) Schematic illustration of the DNA sensor. The sensor is composed of a Cl strand that folds into a hairpin structure and contains an internal 6-FAM “F” and a 3′-BHQ1 “Q” moiety. The Cl is hybridized to a L strand with a 5′-OH end. The cleavage site is indicated by an arrow. (B) Flow chart of the anticipated reaction of hTopI with the DNA sensor. Cleavage results in release of the BHQ1 shifting the sensor to the ON state. Subsequently, ligation of the L strand releases hTopI and leaves the enzyme ready for another round of catalysis. The position of the fluorephore-quencher pair resulted in a quenching efficiency of approximately 80% (data not shown). (C) Gel picture showing the products resulting from incubating the DNA sensor with hTopI followed by trypsin- (lane 2) or proteinase K digestion (lane 3) or no protease treatment (lane 4). Lane 1 shows the gel-electrophoretic mobility of the unreacted sensor. The lengths (in bases) of the substrate and the upper product are indicted to the left of the gel-picture.

Mentions: The sensor was prepared by incubating 50 pmol L strand and 25 pmol Cl strand in 1× TopI-buffer (10 mM Tris-HCl pH 7.5, 5 mM CaCl2, 5 mM MgCl2 and 0.1 mM DTT) for 5 min at 75 °C and cooled to room temperature. The reason for adding a surplus of L strand was to ensure that the more expensive fluorophore-quencher coupled Cl strands was hybridized with an L strand thus forming the sensor. Increasing the relative concentration of the L strand did not significantly affect the reactively of the sensor while decreasing the relative concentration of the L strand reduced the reactivity of the sensor as expected, since the effective concentration of fully annealed and active sensor decreased (data not shown). Subsequently, 600 fmol of purified hTopI enzyme was incubated with the sensor in a total volume of 20 μL containing 1× TopI buffer for 30 min at 37 °C. All reactions were terminated by the addition of SDS to a final concentration of 0.1% (w/v) and the samples precipitated by the addition of 300 mM NaCl and 3 volumes of 96% EtOH. Following precipitation the samples were redissolved in 1× TE-buffer and either left untreated, or digested with 1 mg/mL proteinase K or 0.1 mg/mL trypsin as indicated in Figure 1(C) following standard protocols. The samples were analyzed in a 12% denaturing polyacrylamide gel essentially as described by Christiansen et al. [26].


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) Schematic illustration of the DNA sensor. The sensor is composed of a Cl strand that folds into a hairpin structure and contains an internal 6-FAM “F” and a 3′-BHQ1 “Q” moiety. The Cl is hybridized to a L strand with a 5′-OH end. The cleavage site is indicated by an arrow. (B) Flow chart of the anticipated reaction of hTopI with the DNA sensor. Cleavage results in release of the BHQ1 shifting the sensor to the ON state. Subsequently, ligation of the L strand releases hTopI and leaves the enzyme ready for another round of catalysis. The position of the fluorephore-quencher pair resulted in a quenching efficiency of approximately 80% (data not shown). (C) Gel picture showing the products resulting from incubating the DNA sensor with hTopI followed by trypsin- (lane 2) or proteinase K digestion (lane 3) or no protease treatment (lane 4). Lane 1 shows the gel-electrophoretic mobility of the unreacted sensor. The lengths (in bases) of the substrate and the upper product are indicted to the left of the gel-picture.
© Copyright Policy
Related In: Results  -  Collection

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

f1-sensors-13-04017: (A) Schematic illustration of the DNA sensor. The sensor is composed of a Cl strand that folds into a hairpin structure and contains an internal 6-FAM “F” and a 3′-BHQ1 “Q” moiety. The Cl is hybridized to a L strand with a 5′-OH end. The cleavage site is indicated by an arrow. (B) Flow chart of the anticipated reaction of hTopI with the DNA sensor. Cleavage results in release of the BHQ1 shifting the sensor to the ON state. Subsequently, ligation of the L strand releases hTopI and leaves the enzyme ready for another round of catalysis. The position of the fluorephore-quencher pair resulted in a quenching efficiency of approximately 80% (data not shown). (C) Gel picture showing the products resulting from incubating the DNA sensor with hTopI followed by trypsin- (lane 2) or proteinase K digestion (lane 3) or no protease treatment (lane 4). Lane 1 shows the gel-electrophoretic mobility of the unreacted sensor. The lengths (in bases) of the substrate and the upper product are indicted to the left of the gel-picture.
Mentions: The sensor was prepared by incubating 50 pmol L strand and 25 pmol Cl strand in 1× TopI-buffer (10 mM Tris-HCl pH 7.5, 5 mM CaCl2, 5 mM MgCl2 and 0.1 mM DTT) for 5 min at 75 °C and cooled to room temperature. The reason for adding a surplus of L strand was to ensure that the more expensive fluorophore-quencher coupled Cl strands was hybridized with an L strand thus forming the sensor. Increasing the relative concentration of the L strand did not significantly affect the reactively of the sensor while decreasing the relative concentration of the L strand reduced the reactivity of the sensor as expected, since the effective concentration of fully annealed and active sensor decreased (data not shown). Subsequently, 600 fmol of purified hTopI enzyme was incubated with the sensor in a total volume of 20 μL containing 1× TopI buffer for 30 min at 37 °C. All reactions were terminated by the addition of SDS to a final concentration of 0.1% (w/v) and the samples precipitated by the addition of 300 mM NaCl and 3 volumes of 96% EtOH. Following precipitation the samples were redissolved in 1× TE-buffer and either left untreated, or digested with 1 mg/mL proteinase K or 0.1 mg/mL trypsin as indicated in Figure 1(C) following standard protocols. The samples were analyzed in a 12% denaturing polyacrylamide gel essentially as described by Christiansen et al. [26].

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