UsiRNAs are duplex siRNAs that are modified with non-nucleotide acyclic monomers termed unlocked nucleobase analogs (UNA), and when appropriate, may also contain conformationally restricted nucleotides (CRN). UsiRNAs are designed to enter the RNAi pathway via Dicer enzyme or directly into RISC. UsiRNAs are fully recognized by the RNAi machinery and provide for potent RNAi activity. UsiRNAs are a type of siRNA that can minimize the potential for off-target effects by either strand of the siRNA duplex. UsiRNAs can escape the surveillance mechanisms associated with cytokine induction, and the modifications in UsiRNAs provide protection from nuclease degradation.
UNAs and CRNs in UsiRNAs
Unlocked nucleobase analogs (UNA) are distinct entities, as the bond between two adjacent carbon atoms that form the ribose portion of RNA is absent (see figure below). UNAs are considered to be conformationally flexible. In contrast, conformationally restricted nucleotides (CRN) are novel nucleoside analogs in which the ribose portion is locked into a rigid conformation by a small chemical linker (see figure below).
When included in an siRNA, UNA and CRN monomers enable Marina Biotech to tailor key characteristics to impart greater specificity and activity to the UsiRNA construct. These include:
- UNAs prevent passenger strand participation in RNAi, thus lowering the potential for off-target effects
- UNAs increase the specificity of the guide strand for its intended target mRNA by eliminating microRNA-like off-target effects
- UNA and CRN provide resistance to nuclease degradation
- UNA and CRN decrease the potential for a cytokine response
- CRNs impart the highly stable A-form of RNA to the duplex, resulting in increased thermal stability
In addition, CRN substitution into single-stranded oligonucleotides affords unprecedented stability of single-stranded RNA thus enabling development of novel RNA-based therapeutics that function outside of the RNAi mechanism.