An in solution assay for interrogation of affinity and rational minimer design for small molecule-binding aptamers.
Aptamers are short single-stranded oligonucleotides that fold into unique three-dimensional structures, facilitating selective and high affinity binding to their cognate targets. It is not well understood how aptamer-target interactions affect regions of structure in an aptamer, particularly for small molecule targets where binding is often not accompanied by a dramatic change in structure. The DNase I footprinting assay is a classical molecular biology technique for studying DNA-protein interactions. The simplest application of this method permits identification of protein binding where DNase I digestion is inhibited. Here, we describe a novel variation of the classical DNase I assay to study aptamer-small molecule interactions. Given that DNase I preferentially cleaves duplex DNA over single-stranded DNA, we are able to identify regions of aptamer structure that are affected by small molecule target binding. Importantly, our method allows us to quantify these subtle effects, providing an in solution measurement of aptamer-target affinity. We applied this method to study aptamers that bind to the mycotoxin fumonisin B1, allowing the first identification of high affinity putative minimers for this important food contaminant. We confirmed the binding affinity of these minimers using a magnetic bead binding assay.
Chemistry Department, Carleton University, 1125 Colonel By Drive, Ottawa, ON, Canada K1S 5B6. maria.derosa@carleton.ca.
Nadine R
NR
Frost
Maureen
M
McKeague
Darren
D
Falcioni
Maria C
MC
DeRosa
D052157:Aptamers, Nucleotide / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D003850:Deoxyribonuclease I / Q000378:metabolism; D015195:Drug Design; D059346:Magnets / Q000737:chemistry; D008863:Microspheres; D054852:Small Molecule Libraries / Q000378:metabolism*; D012996:Solutions; D013696:Temperature
D052157:Aptamers, Nucleotide; D054852:Small Molecule Libraries; D012996:Solutions; D003850:Deoxyribonuclease I