| Abstract [eng] |
The development of next-generation sequencing (NGS) was a revolutionary milestone in genome analysis. All current sequencing platforms require nucleic acid pre-processing to generate library suitable for sequencing. However, the workflow is a multistep procedure having several drawbacks and limiting steps (e.g., enzymatic ligation of adapters). Consequently, novel library preparation techniques need to be developed to improve library preparation efficiency and simplify the workflow. Within this work we present the design and synthesis and applications of oligonucleotide-tethered 2’-deoxynucleotides and 2′,3′-dideoxynucleotide terminators ((d)dONNTPs) bearing universal priming sites attached to the nucleobase. Efficient, universal, and scalable synthetic strategy for nucleobase modified nucleotides was developed. The optimized (d)dONNTPs synthesis and purification methodologies enabled to obtain these complex molecules with necessary qualities for enzymatic processes. We showed that even though their structure possesses bulky oligonucleotide label, the linker design empowers both the enzymatic incorporation and read-through. The biocompatibility of five different artificial backbones of nucleic acids was proven, with the single cycle read-through efficiency of 75%. Utilization of ddONNTPs substantially simplified NGS library preparation workflow for high-throughput sequencing. The technology was shown to be applicable for DNA and cDNA library preparation. |
