Department of Chemical and Biomolecular Engineering, Polytechnic Institute of New York University, Brooklyn, NY 11201, USA.
Anal Biochem. 2013 Mar 15;434(2):207-14. doi: 10.1016/j.ab.2012.12.001. Epub 2012 Dec 12.
A principal challenge in microarray experiments is to facilitate hybridization between probe strands on the array with complementary target strands from solution while suppressing any competing interactions that the probes and targets may experience. Synthetic DNA analogs, whose hybridization to targets can exhibit qualitatively different dependence on experimental conditions than for nucleic acid probes, open up an attractive alternative for improving selectivity of array hybridization. Morpholinos (MOs), a class of uncharged DNA analogs, are investigated as microarray probes instead of DNA. MO microarrays were fabricated by contact printing of amino-modified probes onto aldehyde slides. In addition to covalent immobilization, MOs were found to efficiently immobilize through physical adsorption; such physically adsorbed probes could be removed by post-printing washes with surfactant solutions. Hybridization of double-stranded DNA targets to MO microarrays revealed a hybridization maximum at intermediate ionic strengths. The decline in hybridization at lower ionic strengths was attributed to an electrostatic barrier accumulated from hybridized DNA targets, whereas at higher ionic strengths it was attributed to stabilization of target secondary structure in solution. These trends, which illustrate ionic strength tuning of forming on-array relative to solution secondary structure, were supported by a stability analysis of MO/DNA and DNA/DNA duplexes in solution.
微阵列实验中的一个主要挑战是促进阵列上的探针与溶液中互补的靶标链之间的杂交，同时抑制探针和靶标可能经历的任何竞争相互作用。与核酸探针相比，其与靶标杂交的合成 DNA 类似物在实验条件下表现出定性不同的依赖性，为提高阵列杂交的选择性提供了有吸引力的替代方案。拟似物 (MOs) 是一类不带电荷的 DNA 类似物，被研究用作微阵列探针而不是 DNA。MO 微阵列通过将氨基修饰的探针接触印刷到醛基玻片上来制造。除了共价固定化之外，还发现 MOs 通过物理吸附有效地固定；通过用表面活性剂溶液进行打印后清洗可以去除这种物理吸附的探针。双链 DNA 靶标与 MO 微阵列的杂交在中间离子强度下显示出杂交最大值。在较低离子强度下杂交的下降归因于杂交 DNA 靶标累积的静电势垒，而在较高离子强度下则归因于溶液中靶标二级结构的稳定。这些趋势说明了在溶液中形成的相对离子强度对形成的影响，通过对溶液中二聚体中 MO/DNA 和 DNA/DNA 的稳定性分析得到了支持。
