Hybridization and enzymatic extension of Au nanoparticle-bound oligonucleotides
Hybridization and enzymatic extension of Au nanoparticle-bound oligonucleotides
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DOI：
10.1021/ja0177915
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发表时间：
2002-06-26
影响因子：
15
通讯作者：
Keating, CD
中科院分区：
文献类型：
--
作者：
Peña, SRN;Raina, S;Keating, CD
我们研究了空间位阻效应对寡核苷酸与12 nm胶体Au颗粒结合的杂交和酶促延伸的影响。在这些实验中，纳米颗粒结合的12-mer序列与其溶液相12-mer互补物或88-mer模板序列杂交。颗粒结合的寡核苷酸用作酶促延伸反应的引物，其中通过DNA聚合酶的作用实现核苷酸的共价掺入以形成模板的互补物。引物通过连接在5'端的-C6 H12 SH、-C12 H24 SH和-TTACAATC（6）H（12）SH接头连接。通过用（5 ′）HSC（6）H（12）AAA AAA（3 ′）稀释来改变纳米颗粒上的引物覆盖度。杂交效率被确定为接头长度、引物覆盖度、补体长度（12聚体与88聚体）和引物：补体浓度比的函数。在所有情况下，88聚体的杂交效率低于12聚体。低的引物表面覆盖率，更大的颗粒引物分离，和更高的引物：补体比例导致最佳杂交。12聚体和88聚体的杂交效率分别高达98%和75%。在所有测试条件下观察到颗粒结合引物的酶促延伸;然而，反应效率受到接头长度和引物覆盖度的强烈影响。由最长的接头连接的引物的延伸与溶液相反应一样有效。
We have investigated the impact of steric effects on the hybridization and enzymatic extension of oligonuclectides bound to 12-nm colloidal Au particles. In these experiments, a nanoparticle-bound 12-mer sequence is hybridized either to its solution phase 12-mer complement or to an 88-mer template sequence. The particle-bound oligonucleotide serves as a primer for enzymatic extension reactions, in which covalent incorporation of nucleotides to form the complement of the template is achieved by the action of DNA polymerase. Primers were attached via-C6H12SH,-C12H24SH, and -TTACAATC(6)H(12)SH linkers attached at the 5' end. Primer coverage on the nanoparticles was varied by dilution with (5')HSC(6)H(12)AAA AAA(3'). Hybridization efficiencies were determined as a function of linker length, primer coverage, complement length (12-mer vs 88-mer), and primer:complement concentration ratio. In all cases, hybridization for the 88-mer was less efficient than for the 12-mer. Low primer surface coverages, greater particle-primer separation, and higher primer:complement ratios led to optimal hybridization. Hybridization efficiencies as high as 98% and 75% were observed for the 12-mer and 88-mer, respectively. Enzymatic extension of particle-bound primers was observed under all conditions tested; however, the efficiency of the reaction was strongly affected by linker length and primer coverage. Extension of primers attached by the longest linker was as efficient as the solution-phase reaction.