科研之友
微信
新浪微博
Facebook
分享链接
摘要
recently reported that one bivalent MORF (phosphorodiamidate morpholino, a DNA analogue oligomer) exhibited affinity enhancement (ratio of bivalent to monovalent equilibrium constants for binding) to immobilized complementary DNA (cDNA) by surface plasmon resonance (SPR). Because bivalent effectors using oligomers are easily synthesized with molecular spacing between binding sites, an important determinant of binding, adjustable simply by selecting linkers of different dimensions and/ or lengthening or shortening the oligomer chain length, they may have advantages over existing bivalent effectors. We synthesized four bivalent MORFs with different dimensions between binding sites and measured binding affinities and affinity enhancement by SPR. An 18 mer (MORF18) was made bivalent by dimerizing both with disuccinimidyl suberate (DSS) and disuccinimidyl glutarate (DSG) linkers. By again using DSS but adding seven nonbinding adenine bases and by eliminating six binding bases, a total of four bivalent effectors, DSS-MORF12, DSG-MORF18, DSS-MORF18, and DSS-MORF25, were prepared with two different hybridization affinities (i.e. MORF12 and MORF18/ 25) and three different spacings (i.e. 20, 25, and 100 angstrom) between binding sites. The biotinylated cDNA was immobilized on a sensor chip at 500 and 100 RU coating densities providing an average cDNA separation of 25 and 80 A. As expected, bimolecular binding dominated monomolecular binding in all cases, especially at lower MORF effector concentrations and at higher coating densities. All bivalent MORFs showed equilibrium constants superior to their monovalent form and therefore affinity enhancement. DSS-MORF25 showed the highest equilibrium constant for bimolecular binding presumably because of its larger separation between binding sites. Nevertheless, DSS-MORF12 showed the largest affinity enhancement of almost 3 orders of magnitude presumably because the shorter chain lowered the equilibrium constant for monomolecular binding. We have shown that bivalent effectors may be easily synthesized using MORF The results provide further evidence that the use of bivalent effectors can greatly improve MORF pretargeting and, finally, that bivalent MORFs with reduced equilibrium constants may actually exhibit higher affinity enhancement.
- 出版日期2005-10
