Kinetic Analysis of Site-specific Photoaptamer–Protein Cross-linking

Abstract

ssDNA oligonucleotides containing bromodeoxyuridine, BrdU-photoaptamers, are rapidly emerging as specific protein capture reagents in protein microarray technologies. A mathematical model for the kinetic analysis of photoaptamer–protein photocross-linking reactions is presented. The model is based on specific aptamer/protein binding followed by laser excitation that can lead to either covalent cross-linking of the photoaptamer and protein in the complex or irreversible photodamage to the aptamer. Two distinct kinetic regimes, (1) frozen and (2) rapid equilibrium, are developed analytically to model binding kinetics between laser pulses. The models are used to characterize the photocross-linking between three photoaptamers and their cognate protein targets; photoaptamers 0650 and 0615 cross-link human basic fibroblast growth factor and 0518 cross-links HIV MN envelope glycoprotein. Data for cross-linking reaction yields as a function of both laser energy dose and target protein concentration are analyzed for affinity constants and cross-link reaction rates. The binding dissociation constants derived from the cross-linking data are in good accord with independent measurements; the rapid equilibrium model appears to produce results more consistent with the experimental observations, although there is significant overlap between the two models for most conditions explored here. The rate of photodamage for 0615 and 0518 is 3.5 and 2.5 times that of the specific cross-link, giving low maximum reaction yields of ∼20% and ∼30%, whereas 0650 cross-links with a rate over five times higher than its photodamage rate and has a maximum reaction yield exceeding 80%. Quantum yields for the three systems are estimated from the data; photoaptamer 0650 has a reasonably high quantum yield of ∼0.2 for protein cross-linking, while 0518 and 0615 have quantum yields of 0.07 and 0.02. The work presented here provides a useful set of metrics that allow for refinement of photoaptamer properties.