Site-specifically arraying small molecules or proteins on DNA using an expanded genetic alphabet

摘要

We have developed a class of replicable unnatural DNA base pairs formed between d>5SICS and either d>MMO2, d>DMO, or d>NaM. To explore the use of these pairs to produce site-specifically labeled DNA, we report the synthesis of a variety of derivatives bearing propynyl groups, an analysis of their polymerase-mediated replication, and subsequent site-specific modification of the amplified DNA via Click chemistry. We find that with the d>5SICS scaffold, a propynyl ether linker is accommodated better than its aliphatic analog, but not as well as the protected propargyl amine linker explored previously. We also find that with the d>MMO2 and d>DMO analogs, the d>MMO2 position para to the glycosidic linkage is best suited for linker attachment, and that while aliphatic and ether-based linkers are similarly accommodated, the direct attachment of an ethynyl group to the nucleobase core is most well tolerated. To demonstrate the utility of these analogs, a variety of them are used to site-selectively attach a biotin tag to the amplified DNA. Finally, we use d>5SICS^CO-d>NaM to couple one or two proteins to amplified DNA, with the double labeled product visualized by atomic force microscopy. The ability to encode the spatial relationships of arrayed molecules in PCR amplifiable DNA should have important applications, ranging from SELEX with functionalities not naturally present in DNA to the production, and perhaps “evolution” of nanomaterials.