The modification of DNA with indole-linked nucleotides alters its sensitivity to enzymatic cleavage

Abstract

We describe the synthesis of C-5 indole-tagged pyrimidine and C-8 indole-tagged purine nucleoside phosphoramidites and their incorporation into double-stranded DNA 15 base pairs in length. Of the 23 sequence modifications tested, two induced the DNA duplex to adopt a Z-like left-handed conformation under physiological salt conditions, bypassing the specific sequences typically required for a left-handed Z-DNA structure. The impact of these modifications varied with the linker type: flexible propyl linkers exhibited distinct effects compared to rigid propargyl linkers. Notably, modifications positioned directly on or near a restriction site emphasized the pivotal role of linker rigidity in controlling DNA conformation. Specifically, the conformational change induced by the flexible linker impacted nuclease and restriction endonuclease cleavage, reducing sequence specificity. In contrast, the rigid linker suppressed this effect. Furthermore, our findings indicate that nucleic acid duplexes modified with indole-linked nucleotides using a flexible propyl linker have a pronounced tendency to form BZ or Z-like regions in longer DNA sequences. A higher density of modifications may even induce a full Z-like conformation throughout the duplex. These modified nucleotides hold potential for the development of novel antisense therapeutics and introducing valuable tools for in vitro screening of small molecules targeting distorted B-DNA, BZ-DNA, and Z-DNA structures.