Oligonukleotidais modifikuotų nukleotidų sintezė ir panaudojimas

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

Sensitive and accurate analysis of gene expression signatures enabled by oligonucleotide-labelled cDNA

High-throughput RNA sequencing offers a comprehensive analysis of transcriptome complexity origi-nated from regulatory events, such as differential gene expression, alternative polyadenylation and others, and allows the increase in diagnostic capacity and precision. For gene expression profiling applications that do not specifically require information on alternative splicing events, the mRNA 3′ termini counting approach is a cost-effective alternative to whole transcriptome sequencing. Here, we report MTAS-seq (mRNA sequencing via terminator-assisted synthesis) – a novel RNA-seq library pre-paration method directed towards mRNA 3′ termini. We demonstrate the specific enrichment for 3′- terminal regions by simple and quick single-tube protocol with built-in molecular barcoding to enable accurate estimation of transcript abundance. To achieve that, we synthesized oligonucleotide-modified dideoxynucleotides which enable the generation of cDNA libraries at the reverse transcription step. We validated the performance of MTAS-seq on well-characterized reference bulk RNA and further tested it with eukaryotic cell lysates

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

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 (ddONNTP) 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 ddONNTP 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 ddONNTP 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

Fusion sequencing via terminator‐assisted synthesis (FTAS‐seq) identifies TMPRSS2 fusion partners in prostate cancer

Genetic rearrangements that fuse an androgen‐regulated promoter area with a protein‐coding portion of an originally androgen‐unaffected gene are frequent in prostate cancer, with the fusion between transmembrane serine protease 2 (TMPRSS2) and ETS transcription factor ERG (ERG) (TMPRSS2‐ERG fusion) being the most prevalent. Conventional hybridization‐ or amplification‐based methods can test for the presence of expected gene fusions, but the exploratory analysis of currently unknown fusion partners is often cost‐prohibitive. Here, we developed an innovative next‐generation sequencing (NGS)‐based approach for gene fusion analysis termed fusion sequencing via terminator‐assisted synthesis (FTAS‐seq). FTAS‐seq can be used to enrich the gene of interest while simultaneously profiling the whole spectrum of its 3′‐terminal fusion partners. Using this novel semi‐targeted RNA‐sequencing technique, we were able to identify 11 previously uncharacterized TMPRSS2 fusion partners and capture a range of TMPRSS2‐ERG isoforms. We tested the performance of FTAS‐seq with well‐characterized prostate cancer cell lines and utilized the technique for the analysis of patient RNA samples. FTAS‐seq chemistry combined with appropriate primer panels holds great potential as a tool for biomarker discovery that can support the development of personalized cancer therapies