Title: Chemical synthesis and non-chromatographic purification of long RNA oligonucleotides containing naturally occurring modifications. ABSTRACT: Solid phase synthesis of RNA is an important genomic tool, which offers precise control over the oligonucleotide sequence and offers an opportunity for site-specific incorporation of RNA modifications, fluorescent labels and biochemical tags. Discoveries of the twenty-first century created a strong need for a robust synthesis of RNA strands, that are 100-200 nucleotides (-nt) in length. A major limitation of otherwise highly optimized process is purification, which is notoriously difficult, labor intensive and requires expensive HPLC instrumentation. As the result, solid phase synthesis of long oligonucleotides containing RNA modifications is rarely attempted. This technology-development proposal aims to address this limitation by developing a non- chromatographic RNA purification method which will be 10-times faster and 2-orders of magnitude cheaper and will allow isolation of strands that are 100-200-nt long in good yield and 98% purity. The proposed purification strategy is based on bio-orthogonal inverse electron demand Diels-Alder (IEDDA) chemistry between trans-cyclooctene (TCO) and tetrazine (Tz) that allows to selectively tag and purify structurally complex and increasing long RNA strands from the failure strands that accrue during solid phase synthesis. TCO and Tz are highly selective for each other and have minimal cross-reactivity with other functional groups found in RNA. The bio-orthogonal click chemistry is highly efficient, even at very low concentrations of TCO and Tz. During preliminary studies we have shown that our strategy allows efficient synthesis and purification of 76-nt long tRNA and 101-nt long sgRNA with yields that were 10-times higher than conventional purification methods. During the proposed research program, we aim to improve a number of important elements of our design to bring the overall process to under 7 hrs, further improve the overall yield and purity of the isolated RNA. In Aim 1, we propose to expedite the process by developing a new photolabile linker that will allow fast photocleavage using visible light. To improve purity and yield of isolated RNA, we propose to optimize the solid phase synthesis procedure to ensure that all failure sequences are fully capped during each synthetic cycle. The optimized process will be applied to increasingly longer RNA strands, from 76-nt tRNA to 188-nt long U2 snRNA. In Aim 2, we plan to illustrate the power of our technology by synthesizing a 144-nt long artificial mRNA, containing m1A and m6A modifications near the start codon. This will be the longest reported oligonucleotide containing RNA modifications. The artificial mRNA will be utilized to investigate the impact of RNA modifications on the rate of in vitro translation. We plan to work with NHGRI technology development team to make the proposed technology widely av...