Conjugation of Duplexed siRNN Oligonucleotides with DD-HyNic Peptides for Cellular Delivery of RNAi Triggers   

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Original research article

A brief version of this protocol appeared in:
Nature Biotechnology
Dec 2014


Despite the great promise that short interfering RNA (siRNA) induced RNAi responses hold as a therapeutic modality, due to their size (~15 kDa) and high negative charge (Bumcrot et al., 2006), siRNAs have no bioavailability and require a delivery agent to enter cells (Figure 1). TAT peptide transduction domain (PTD) has been developed as an agent that mediates cellular delivery of macromolecular therapeutics that otherwise lack bioavailability, making it a tantalizing candidate for siRNA delivery (Farkhani et al., 2014). Unfortunately, when conjugated to TAT PTD, the presence of 40 negative phosphodiester backbone charges on siRNA neutralizes the cationic PTD resulting in aggregation and poor cellular delivery (Meade and Dowdy, 2007). In light of this, we synthesized a neutral RNAi trigger, termed siRiboNucleic Neutrals, for conjugation to TAT PTD (Meade et al., 2014). In brief, the negatively charged phosphodiester backbone was neutralized by synthesis with bio-reversible phosphotriester protecting groups which are specifically converted into charged phosphodiester bonds inside of cells by the action of cytoplasmic restricted thioesterases resulting in a wild type siRNA that can induce RNAi responses. Here we describe the conjugation and cellular delivery of siRNN oligonucleotides with TAT PTD delivery domain (DD) HyNic peptides.

Keywords: siRNA, siRNN, Phosphotriester, Peptide Transduction Domain, Oligonucleotide conjugation

Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
How to cite: Hamil, A. S., Gogoi, K. and Dowdy, S. F. (2016). Conjugation of Duplexed siRNN Oligonucleotides with DD-HyNic Peptides for Cellular Delivery of RNAi Triggers. Bio-protocol 6(7): e1782. DOI: 10.21769/BioProtoc.1782.

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