RNA interference-based manipulation of gene expression is a highly versatile tool available to insect scientists. In some cases direct injection of large amounts of long dsRNA will suffice. In other cases, not so much. Combining siRNA with some kind of carrier could improve gene silencing but siRNA has poor charge density making its association with charged carriers tenuous and weak. The RNAi machinery can accommodate a number of unconventional RNA triggers that consist of RNA with different structures and this can be exploited to improve charge density, improve interactions with charged carriers and improved silencing.
Sajeesh et al (2014) in the Journal of Controlled Release report on their use of ‘tripodal RNA’ in conjunction with linear polyethyleneimine (PEI) and galactose-modified PEI to effectively silence genes. Linear PEI is the preferred choice for RNA delivery.
Tripodal RNA (tiRNA) is a non-linear form of interfering RNA that appears to act using a Dicer-independent pathway. Tripodal RNAs used by Sajeesh et al. were 26, 32 and 40nt long with most experiments performed with tiRNA-32 because, according to them, they were easier to synthesize. What is particularly interesting about this kind of RNAi trigger is the fact that each tiRNA can ‘hit’ three targets. Of course, those targets can be within the same or different genes.
When tiRNA was complexed with linear PEI such that the positive to negative charge ratio was 5 or 10 Sajeesh et al observed a 3-fold increase in the uptake of the complex compared to when tiRNA was replaced with siRNA. tiRNA appeared to result in a significant increase in the uptake of the RNAi trigger.
Not only was uptake improved but so was gene silencing in vitro. So this is an interesting and easy alternative to what might be considered in lieu of the standard approach in insect science, injecting massive quantities of long dsRNA, particularly when observed silencing responses are less than desired.
Sajeesh et al (2014) were really interested in delivering RNAi triggers specifically to liver cells in vivo. Their strategy was to add GAL (aminophenyl β-D-galatopyranoside) to PEI and use this derivatized carrier for tiRNA. While Sajeesh et al do not report on experiments performed in vivo they do report a large increase in the cellular uptake of tiRNA complexes into liver cells in vitro. So overall these results were encouraging.
Insect scientists struggling with getting good silencing in vivo might consider this relatively simple alternative. Sajeesh et al (2014) report their methods with sufficient detail so others should be able to produce their own tiRNA, tiRNA-PEI and tiRNA-PEI-Gal complexes.
Sajeesh S, Lee TY, Kim JK, Son DS, Hong SW, Kim S, Yun WS, Kim S, Chang C, Li C, Lee D-k (2014) Efficient intracellular delivery and multiple-target gene silencing triggered by tripodal RNA based nanoparticles: A promising approach in liver-specific RNAi delivery. J Control Release 196: 28-36 http://dx.doi.org/10.1016/j.jconrel.2014.09.016