Limits...
A 96-well DNase I footprinting screen for drug-DNA interactions.

Ellis T, Evans DA, Martin CR, Hartley JA - Nucleic Acids Res. (2007)

Bottom Line: A semi-automated analysis system has also been developed to present footprinting data as an estimate of the binding affinity of each tested compound to any base pair in the assessed DNA sequence, giving an intuitive 'one compound-one line' scheme.Here, we demonstrate the screening capabilities of the 96-well assay and the subsequent data analysis using a series of six pyrrolobenzodiazepine-polypyrrole compounds and human Topoisomerase II alpha promoter DNA.The dramatic increase in throughput, quantified data and decreased handling time allow, for the first time, DNase I footprinting to be used as a screening tool to assess DNA-binding agents.

View Article: PubMed Central - PubMed

Affiliation: Spirogen Ltd, London Bioscience Innovation Centre, London, UK.

ABSTRACT
The established protocol for DNase I footprinting has been modified to allow multiple parallel reactions to be rapidly performed in 96-well microtitre plates. By scrutinizing every aspect of the traditional method and making appropriate modifications it has been possible to considerably reduce the time, risk of sample loss and complexity of footprinting, whilst dramatically increasing the yield of data (30-fold). A semi-automated analysis system has also been developed to present footprinting data as an estimate of the binding affinity of each tested compound to any base pair in the assessed DNA sequence, giving an intuitive 'one compound-one line' scheme. Here, we demonstrate the screening capabilities of the 96-well assay and the subsequent data analysis using a series of six pyrrolobenzodiazepine-polypyrrole compounds and human Topoisomerase II alpha promoter DNA. The dramatic increase in throughput, quantified data and decreased handling time allow, for the first time, DNase I footprinting to be used as a screening tool to assess DNA-binding agents.

Show MeSH
A comparison of footprint profiles of compounds 1–6 on the proximal region of the TOPOIIα promoter. Single footprint profiles were program-generated starting with the IR footprint gel image shown in Figure 3A. Where no footprinting is seen, the sequence has been given an arbitrary score of −3.9 (log 125 μM). The DNA sequence is given in Supplementary Figure 1.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC1919508&req=5

Figure 4: A comparison of footprint profiles of compounds 1–6 on the proximal region of the TOPOIIα promoter. Single footprint profiles were program-generated starting with the IR footprint gel image shown in Figure 3A. Where no footprinting is seen, the sequence has been given an arbitrary score of −3.9 (log 125 μM). The DNA sequence is given in Supplementary Figure 1.

Mentions: Our initial intention was to use quantitative DNase I footprint titrations to determine an association constant (Ka) for every single nucleotide by fitting background-corrected band intensities to the Hill equation (28). Although this approach was successful at many binding sites, its universal use was not feasible as band intensity decreases were insufficient in regions that were not footprinted. Additionally, the requirement to model thousands of curves required more computing power and data manipulation than was practicable. Next, we looked at a simpler approach that can be adopted when the lowest concentrations at which footprints are observed are greater than the concentration of DNA in the samples (8). In this method, the ligand dissociation constant is effectively given by the ligand concentration required to produce a 50% decrease in band intensity (29). In the screen described here, observable decreases in band intensity due to binding are almost always greater than 50% due to the 5-fold concentration difference between sample lanes. Subsequently, it was possible to design a simple program to determine, for every nucleotide position, the ligand concentration at which a significant band intensity decrease is observed compared to the control. This value, referred to as the ‘score’ (log concentration), gives a good approximation of the dissociation constant by effectively estimating the C50 value. The program uses the differential cleavage data and a user-defined differential cleavage cut-off value to determine a ‘score’ for every nucleotide position by reporting the lowest concentration at which the differential cleavage value at that base is lower than the cut-off value. The result is a ‘score’ profile across the tested concentration range that describes the apparent affinity of a compound to each position in the entire DNA sequence tested. Positions with no band intensity decrease over the tested range are assigned a ‘score’ equivalent to binding at 125 μM, 5-fold above the maximum tested concentration; which allows them to be easily disregarded. A plot of the ‘score’ profile allows primary binding sites to be distinguished from secondary and tertiary binding sites, as the primary sites are bound with a higher approximate association constant and thus receive a lower ‘score’ value (Figure 3C). In addition, the DNA-binding properties of multiple compounds can readily be compared when multiple ‘score’ profiles are displayed together (Figure 4).Figure 4.


A 96-well DNase I footprinting screen for drug-DNA interactions.

Ellis T, Evans DA, Martin CR, Hartley JA - Nucleic Acids Res. (2007)

A comparison of footprint profiles of compounds 1–6 on the proximal region of the TOPOIIα promoter. Single footprint profiles were program-generated starting with the IR footprint gel image shown in Figure 3A. Where no footprinting is seen, the sequence has been given an arbitrary score of −3.9 (log 125 μM). The DNA sequence is given in Supplementary Figure 1.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC1919508&req=5

Figure 4: A comparison of footprint profiles of compounds 1–6 on the proximal region of the TOPOIIα promoter. Single footprint profiles were program-generated starting with the IR footprint gel image shown in Figure 3A. Where no footprinting is seen, the sequence has been given an arbitrary score of −3.9 (log 125 μM). The DNA sequence is given in Supplementary Figure 1.
Mentions: Our initial intention was to use quantitative DNase I footprint titrations to determine an association constant (Ka) for every single nucleotide by fitting background-corrected band intensities to the Hill equation (28). Although this approach was successful at many binding sites, its universal use was not feasible as band intensity decreases were insufficient in regions that were not footprinted. Additionally, the requirement to model thousands of curves required more computing power and data manipulation than was practicable. Next, we looked at a simpler approach that can be adopted when the lowest concentrations at which footprints are observed are greater than the concentration of DNA in the samples (8). In this method, the ligand dissociation constant is effectively given by the ligand concentration required to produce a 50% decrease in band intensity (29). In the screen described here, observable decreases in band intensity due to binding are almost always greater than 50% due to the 5-fold concentration difference between sample lanes. Subsequently, it was possible to design a simple program to determine, for every nucleotide position, the ligand concentration at which a significant band intensity decrease is observed compared to the control. This value, referred to as the ‘score’ (log concentration), gives a good approximation of the dissociation constant by effectively estimating the C50 value. The program uses the differential cleavage data and a user-defined differential cleavage cut-off value to determine a ‘score’ for every nucleotide position by reporting the lowest concentration at which the differential cleavage value at that base is lower than the cut-off value. The result is a ‘score’ profile across the tested concentration range that describes the apparent affinity of a compound to each position in the entire DNA sequence tested. Positions with no band intensity decrease over the tested range are assigned a ‘score’ equivalent to binding at 125 μM, 5-fold above the maximum tested concentration; which allows them to be easily disregarded. A plot of the ‘score’ profile allows primary binding sites to be distinguished from secondary and tertiary binding sites, as the primary sites are bound with a higher approximate association constant and thus receive a lower ‘score’ value (Figure 3C). In addition, the DNA-binding properties of multiple compounds can readily be compared when multiple ‘score’ profiles are displayed together (Figure 4).Figure 4.

Bottom Line: A semi-automated analysis system has also been developed to present footprinting data as an estimate of the binding affinity of each tested compound to any base pair in the assessed DNA sequence, giving an intuitive 'one compound-one line' scheme.Here, we demonstrate the screening capabilities of the 96-well assay and the subsequent data analysis using a series of six pyrrolobenzodiazepine-polypyrrole compounds and human Topoisomerase II alpha promoter DNA.The dramatic increase in throughput, quantified data and decreased handling time allow, for the first time, DNase I footprinting to be used as a screening tool to assess DNA-binding agents.

View Article: PubMed Central - PubMed

Affiliation: Spirogen Ltd, London Bioscience Innovation Centre, London, UK.

ABSTRACT
The established protocol for DNase I footprinting has been modified to allow multiple parallel reactions to be rapidly performed in 96-well microtitre plates. By scrutinizing every aspect of the traditional method and making appropriate modifications it has been possible to considerably reduce the time, risk of sample loss and complexity of footprinting, whilst dramatically increasing the yield of data (30-fold). A semi-automated analysis system has also been developed to present footprinting data as an estimate of the binding affinity of each tested compound to any base pair in the assessed DNA sequence, giving an intuitive 'one compound-one line' scheme. Here, we demonstrate the screening capabilities of the 96-well assay and the subsequent data analysis using a series of six pyrrolobenzodiazepine-polypyrrole compounds and human Topoisomerase II alpha promoter DNA. The dramatic increase in throughput, quantified data and decreased handling time allow, for the first time, DNase I footprinting to be used as a screening tool to assess DNA-binding agents.

Show MeSH