Department of Chemistry and Center for Nucleic Acids Science and Technology, Carnegie Mellon University, 4400 Fifth Avenue, Pittsburgh, PA 15213-3890, USA.
Department of Biological Sciences, Carnegie Mellon University, 4400 Fifth Avenue, Pittsburgh, PA 15213-3890, USA.
Molecules. 2020 Feb 21;25(4):970. doi: 10.3390/molecules25040970.
High affinity nucleic acid analogues such as gammaPNA (γPNA) are capable of invading stable secondary and tertiary structures in DNA and RNA targets but are susceptible to off-target binding to mismatch-containing sequences. We introduced a hairpin secondary structure into a γPNA oligomer to enhance hybridization selectivity compared with a hairpin-free analogue. The hairpin structure features a five base PNA mask that covers the proximal five bases of the γPNA probe, leaving an additional five γPNA bases available as a toehold for target hybridization. Surface plasmon resonance experiments demonstrated that the hairpin probe exhibited slower on-rates and faster off-rates (i.e., lower affinity) compared with the linear probe but improved single mismatch discrimination by up to a factor of five, due primarily to slower on-rates for mismatch vs. perfect match targets. The ability to discriminate against single mismatches was also determined in a cell-free mRNA translation assay using a luciferase reporter gene, where the hairpin probe was two-fold more selective than the linear probe. These results validate the hairpin design and present a generalizable approach to improving hybridization selectivity.
高亲和力核酸类似物，如 γPNA（γ 肽核酸），能够侵入 DNA 和 RNA 靶标中的稳定二级和三级结构，但容易与包含错配的序列发生非靶标结合。我们在 γPNA 寡聚物中引入了发夹二级结构，以提高与无发夹类似物相比的杂交选择性。发夹结构具有一个五碱基 PNA 掩蔽物，覆盖 γPNA 探针的近端五个碱基，留下另外五个 γPNA 碱基作为目标杂交的立足点。表面等离子体共振实验表明，与线性探针相比，发夹探针的上样速率较慢，脱附速率较快（即亲和力较低），但单错配区分度提高了五倍，主要是因为错配与完全匹配的靶标相比，上样速率较慢。在使用荧光素酶报告基因的无细胞 mRNA 翻译测定中，也确定了区分单错配的能力，其中发夹探针比线性探针选择性高两倍。这些结果验证了发夹设计，并提出了一种可推广的方法来提高杂交选择性。
