Comparison of Several Linear Fluorophore- and Quencher-Conjugated Oligomer Duplexes for Stability, Fluorescence Quenching, and Kinetics in Vitro and in Vivo in Mice
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- 作者：Surong Zhang ; Guozheng Liu ; Xinrong Liu ; Dongguang Yin ; Shuping Dou ; Jiang He ; Mary Rusckowski ; Donald J. Hnatowich
- 刊名：Bioconjugate Chemistry
- 出版年：2007
- 出版时间：July 2007
- 年：2007
- 卷：18
- 期：4
- 页码：1170 - 1175
- 全文大小：149K
- 年卷期：v.18,no.4(July 2007)
- ISSN：1520-4812
文摘
A useful property of optical imaging is the potential to modulate the detectable signal to improve target/nontargetratios. When administered as a dimer of a fluorophore- and a quencher-conjugated duplex arranged to inhibitfluorescence but designed to dissociate only in the presence of its target, the fluorescence signal should in principleappear only in the target. This laboratory has demonstrated the feasibility of this approach by using a duplexconsisting of a linear oligomer conjugated with Cy5.5 (emitter) hybridized to another linear oligomer conjugatedwith Iowa Black (quencher) in a pretargeting optical study. Now eight duplexes consisting of combinations of 18mer 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 were investigated in vitro and in vivo in mice. Thereafter, mice were implanted in onethigh with MORF/PO Cy 5.5 microspheres and the complementary PS Iowa Black administered iv to measurethe extent and kinetics of duplex formation in the target. While all duplexes were stable in buffer, only theMORF/PO duplexes and possibly all PS containing duplexes were stable in 37 C serum for at least 4 h. Thekinetics of quenching were found to be rapid in vitro, with a 80-90% decrease in Cy5.5 fluorescence immediatelyfollowing formation of a PS/PS homoduplex, and in vivo, with a 27 to 38% decrease in target thigh/nontargetratio within 1 h following administration of the complementary PS Iowa Black complementary DNA but not therandom control DNA to mice implanted with MORF/PO Cy5.5 microspheres. This investigation has providedadditional evidence that Cy5.5 may be efficiently and rapidly quenched by Iowa Black when both are conjugatedto complementary oligomers and that the resulting inhibition of fluorescence is sufficiently persistent for imaging.
C serum. Simultaneously,the kinetics of quenching were investigated in vitro and in vivo in mice. Thereafter, mice were implanted in onethigh with MORF/PO Cy 5.5 microspheres and the complementary PS Iowa Black administered iv to measurethe extent and kinetics of duplex formation in the target. While all duplexes were stable in buffer, only theMORF/PO duplexes and possibly all PS containing duplexes were stable in 37 C serum for at least 4 h. Thekinetics of quenching were found to be rapid in vitro, with a 80-90% decrease in Cy5.5 fluorescence immediatelyfollowing formation of a PS/PS homoduplex, and in vivo, with a 27 to 38% decrease in target thigh/nontargetratio within 1 h following administration of the complementary PS Iowa Black complementary DNA but not therandom control DNA to mice implanted with MORF/PO Cy5.5 microspheres. This investigation has providedadditional evidence that Cy5.5 may be efficiently and rapidly quenched by Iowa Black when both are conjugatedto complementary oligomers and that the resulting inhibition of fluorescence is sufficiently persistent for imaging.