Zhang Surong, Liu Guozheng, Liu Xinrong, Yin Dongguang, Dou Shuping, He Jiang, Rusckowski Mary, Hnatowich Donald J
Department of Radiology, University of Massachusetts Medical School, Worcester, Massachusetts 01655, USA.
Bioconjug Chem. 2007 Jul-Aug;18(4):1170-5. doi: 10.1021/bc070021j. Epub 2007 May 19.
A useful property of optical imaging is the potential to modulate the detectable signal to improve target/nontarget 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. Now eight 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 degrees C serum. Simultaneously, the kinetics of quenching were 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. While all duplexes were stable in buffer, only the MORF/PO duplexes and possibly all PS containing duplexes were stable in 37 degrees C serum for at least 4 h. The kinetics of quenching were found to be rapid in vitro, with a 80-90% decrease in Cy5.5 fluorescence immediately following formation of a PS/PS homoduplex, and in vivo, with a 27 to 38% decrease in target thigh/nontarget ratio within 1 h following administration of the complementary PS Iowa Black complementary DNA but not the random control DNA to mice implanted with MORF/PO Cy5.5 microspheres. This investigation has provided additional evidence that Cy5.5 may be efficiently and rapidly quenched by Iowa Black when both are conjugated to complementary oligomers and that the resulting inhibition of fluorescence is sufficiently persistent for imaging.
光学成像的一个有用特性是有潜力调节可检测信号以提高目标/非目标比率。当作为荧光团和猝灭剂共轭双链体的二聚体给药时，该双链体被设计成抑制荧光，但仅在其目标存在时解离，原则上荧光信号应仅出现在目标中。本实验室在一项预靶向光学研究中，通过使用由与Cy5.5（发射体）共轭的线性寡聚物与与艾奥瓦黑（猝灭剂）共轭的另一个线性寡聚物杂交而成的双链体，证明了这种方法的可行性。现在，对由18聚体线性磷酸二酯（PO）和硫代磷酸酯（PS）DNA以及与Cy5.5（发射体）和艾奥瓦黑（猝灭剂）共轭的吗啉代磷酰胺（MORF）组合而成的八个双链体进行了体外双链体稳定性的各种筛选。基于在37℃血清中的稳定性证据，选择了MORF/PO双链体进行进一步研究。同时，在小鼠体内和体外研究了猝灭动力学。此后，在小鼠的一条大腿中植入MORF/PO Cy 5.5微球，并静脉注射互补的PS艾奥瓦黑，以测量目标中双链体形成的程度和动力学。虽然所有双链体在缓冲液中都稳定，但只有MORF/PO双链体以及可能所有含PS的双链体在37℃血清中至少稳定4小时。发现猝灭动力学在体外很快，在形成PS/PS同型双链体后，Cy5.5荧光立即下降80 - 90%，在体内，在给植入MORF/PO Cy5.5微球的小鼠注射互补的PS艾奥瓦黑互补DNA而非随机对照DNA后1小时内，目标大腿/非目标比率下降27%至38%。这项研究提供了更多证据，表明当Cy5.5和艾奥瓦黑都与互补寡聚物共轭时，Cy5.5可能被艾奥瓦黑有效且快速地猝灭，并且由此产生的荧光抑制对于成像来说足够持久。
