Comparison of several linear fluorophore-and quencher conjugated oligomer duplexes for stability fluorescence quenching and kinetics in vitro and in vivo in mice: Fluorescent DNA duplexes for imaging applications

摘要

A useful property of optical imaging is the potential to modulate the detectable signal to improve targetontarget ratios. When administered as a dimer of a fluorophore- and a quencher-conjugated duplex arranged to inhibit fluorescence but designed to dissociate only in the presence of its target, the fluorescence signal should in principle appear only in the target. This laboratory has demonstrated the feasibility of this approach by using a duplex consisting of a linear oligomer conjugated with Cy5.5 (emitter) hybridized to another linear oligomer conjugated with Iowa Black (quencher) in a pretargeting optical study.MethodsEight duplexes consisting of combinations of 18 mer linear phosphodiester (PO) and phosphorothioate (PS) DNAs and phosphorodiamidate morpholinos (MORFs) conjugated with Cy5.5 (emitter) and Iowa Black (quencher) were variously screened for in vitro duplex stability. The MORF/PO duplex was selected for further study based on evidence of stability in 37 °C serum. Simultaneously, the kinetics of quenching was investigated in vitro and in vivo in mice. Thereafter, mice were implanted in one thigh with MORF/PO Cy 5.5 microspheres and the complementary PS Iowa Black administered IV to measure the extent and kinetics of duplex formation in the target.