Advanced preparation of fragment libraries enabled by oligonucleotide-modified 2′,3′-dideoxynucleotides

Next-generation genome sequencing technologies have revolutionized the life sciences, however all sequencing platforms require nucleic acid pre-processing to generate suitable libraries for sequencing. Here, oligonucleotide-tethered 2 ',3 '-dideoxynucleotide terminators bearing universal priming sites are synthesised and incorporated by DNA polymerases, allowing integration of the fragmentation step into the library preparation workflow while also enabling the obtained fragments to be readily labeled by platform-specific adapters. The ever-growing demand for inexpensive, rapid, and accurate exploration of genomes calls for refinement of existing sequencing techniques. The development of next-generation sequencing (NGS) was a revolutionary milestone in genome analysis. While modified nucleotides already were inherent tools in sequencing and imaging, further modification of nucleotides enabled the expansion into even more diverse applications. Herein we describe the design and synthesis of oligonucleotide-tethered 2 ',3 '-dideoxynucleotide (dd(ON)NTP) terminators bearing universal priming sites attached to the nucleobase, as well as their enzymatic incorporation and performance in read-through assays. In the context of NGS library preparation, the incorporation of dd(ON)NTP fulfills two requirements at once: the fragmentation step is integrated into the workflow and the obtained fragments are readily labeled by platform-specific adapters. DNA polymerases can incorporate dd(ON)NTP nucleotides, as shown by primer extension assays. More importantly, reading through the unnatural linkage during DNA synthesis was demonstrated, with 25-30% efficiency in single-cycle extension.