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Albany 2015:Book of Abstracts

Albany 2015
Conversation 19
June 9-13 2015
©Adenine Press (2012)

Fast Click-Slow Release Strategy Towards HPLC-Free Synthesis of RNA Strands

A general strategy for the purification of bio-polymeric materials, such as RNA strands, synthesized by solid phase synthesis will be described. It is based on a recently developed concept involving bio-orthogonal inverse electron demand Diels-Alder (IEDDA) reaction between trans-cyclooctene and tetrazine, termed 'click-to-release' (Versteegen, Rossin, Hoeve, Janssen, & Robillard, 2013). The unique feature of this system is attachment of a releasable payload at the allylic position of the trans-cyclooctene. After the initial cycloaddition step, the payload is spontaneously released over time. The kinetics of the click and release steps are very different and are optimized through appropriate tetrazine substituent groups. This chemistry allows for efficient immobilization of the trans-cyclooctene derivatized RNA strands using agarose modified with tetrazine. After filtration of the species that did not 'click;, the target strand can be obtained by spontaneous release from the solid support over time. This approach is complementary to other known strategies that facilitate purification of synthetic RNA strands, including biotinylation, polymerization and perfluorination (Beller, & Bannwarth, 2005; Fang, & Bergstrom, 2004; Fang, & Fueangfung, 2010; Fang, Fueangfung, Lin, Zhang, Mai, Bi, & Green, 2011). The presented method is based on IEDDA chemistry which allows for robust purification of RNA under mild conditions due to orthogonality of trans-cyclooctene and tetrazine to the functional groups found in RNA. As a proof-of-principle, the strategy has been applied to the synthesis and purification of the hairpin RNA strand, SL3, which is a known substrate for the HIV nucleocapsid protein, NCp7.

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References
    Beller, C. & Bannwarth, W. (2005). Noncovalent attachment of nucleotides by fluorous-fluorous interactions: application to a simple purification principle for synthetic DNA fragments. Helv. Chim. Acta, 88, 171-179.

    Fang, S. & Bergstrom, D. E. (2004). Reversible 5'-end biotinylation and affinity purification of synthetic RNA. Tetrahedron Lett. 45, 7987-7990.

    Fang, S. & Fueangfung, S. (2010). Scalable synthetic oligodeoxynucleotide purification with use of a catching by polymerization, washing and releasing approach. Org. Lett. 12, 3720-3723.

    Fang, S. Fueangfung, S. Lin, X. Zhang, X. Mai, W. Bi, L. & Green, S. A. (2011). Synthetic oligodeoxynucleotide purification by polymerization of falure sequences. Chem. Commun. 47, 1345-1347.

    Versteegen, R. M. Rossin, R. Hoeve, W. Janssen H. M. & Robillard M. S. (2013). Click to release: instanteneous doxorubicin elimination upon tetrazine ligation. Angew. Chem. Int. Ed. 52, 14112-14116.


Papa Nii Asare Okai
Edgar Agustin
Michael R. Miller
Jia Sheng
Maksim Royzen

Department of Chemistry
University at Albany, SUNY
Albany, NY 12222

Phone: (518) 437-4463
mroyzen@albany.edu