A rapid assay for affinity and kinetics of molecular interactions with nucleic acids.
The Differential Radial Capillary Action of Ligand Assay (DRaCALA) allows detection of protein interactions with low-molecular weight ligands based on separation of the protein-ligand complex by differential capillary action. Here, we present an application of DRaCALA to the study of nucleic acid-protein interactions using the Escherichia coli cyclic AMP receptor protein (CRP). CRP bound in DRaCALA specifically to (32)P-labeled oligonucleotides containing the consensus CRP binding site, but not to oligonucleotides with point mutations known to abrogate binding. Affinity and kinetic studies using DRaCALA yielded a dissociation constant and dissociation rate similar to previously reported values. Because DRaCALA is not subject to ligand size restrictions, whole plasmids with a single CRP-binding site were used as probes, yielding similar results. DNA can also function as an easily labeled carrier molecule for a conjugated ligand. Sequestration of biotinylated nucleic acids by streptavidin allowed nucleic acids to take the place of the protein as the immobile binding partner. Therefore, any molecular interactions involving nucleic acids can be tested. We demonstrate this principle utilizing a bacterial riboswitch that binds cyclic-di-guanosine monophosphate. DRaCALA is a flexible and complementary approach to other biochemical methods for rapid and accurate measurements of affinity and kinetics at near-equilibrium conditions.
Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, MD 20742, USA.
Gregory P
GP
Donaldson
Kevin G
KG
Roelofs
Yiling
Y
Luo
Herman O
HO
Sintim
Vincent T
VT
Lee
D001665:Binding Sites; D006152:Cyclic GMP / Q000031:analogs & derivatives / Q000378:metabolism; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D015342:DNA Probes / Q000737:chemistry; D004268:DNA-Binding Proteins / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000378:metabolism; D007700:Kinetics; D008024:Ligands; D015336:Molecular Probe Techniques; D009696:Nucleic Acids / Q000378:metabolism*; D015345:Oligonucleotide Probes / Q000737:chemistry; D010957:Plasmids / Q000235:genetics; D011953:Receptors, Cyclic AMP / Q000378:metabolism; D058928:Riboswitch
D015342:DNA Probes; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D008024:Ligands; D009696:Nucleic Acids; D015345:Oligonucleotide Probes; D011953:Receptors, Cyclic AMP; D058928:Riboswitch; C047947:cyclic AMP receptor protein, E coli; C062025:bis(3',5')-cyclic diguanylic acid; D004247:DNA; D006152:Cyclic GMP