Activation of different split functionalities on re-association of RNA–DNA hybrids

Abstract

Split-protein systems, an approach that relies on fragmentation of proteins with their further conditional re-association to form functional complexes, are increasingly used for various biomedical applications. This approach offers tight control of the protein functions and improved detection sensitivity. Here we show a similar technique based on a pair of RNA-DNA hybrids that can be generally used for triggering different split functionalities. Individually, each hybrid is inactive but when two cognate hybrids re-associate, different functionalities are triggered inside mammalian cells. As a proof of concept this work is mainly focused on activation of RNA interference; however the release of other functionalities (resonance energy transfer and RNA aptamer) is also shown. Furthermore, in vivo studies demonstrate a significant uptake of the hybrids by tumors together with specific gene silencing. This split-functionality approach presents a new route in the development of “smart” nucleic acids based nanoparticles and switches for various biomedical applications.

De novo design of an RNA tile that self-assembles into a homo-octameric nanoprism

“…Here we expand the design principle to achieve higher structural complexity along this direction. Besides addressing the fundamental challenge in programmed RNA self-assembly, we believe that such a study will increase our capability of rationally designing RNA nanostructures 33,34 to meet the needs of potential applications such as siRNA delivery 5,6 and construction of in vivo RNA machineries 4 . In addition, we have observed a surprising but interesting phenomenon.…”

“…They promise many technological applications, such as drug delivery 5,6 and arranging chemical reactions to produce useful chemicals 7 . Many exciting works have been reported recently along this line 8,9 .…”

RNA Toehold Interactions Initiate Conditional Gene Silencing

“…While each of the approaches above present intrinsic benefits in their design and are able to activate the RNAi pathway in a controllable fashion, both approaches exhibit inherent flaws unique to each design. Split-function nucleic acid hybrids show prolonged persistence in the blood in vivo [4], but require interaction between two exogenous cognate hybrids for RNAi activation after cellular entry. This allows RNAi activation to be temporally controlled by the administration of the second hybrid, however, this mode of activation does not allow for gene silencing in response to a cellular RNA marker.…”

“…Aptamers that specifically bind a particular cell-surface protein have frequently been linked to siRNAs and larger RNA nanoparticles to restrict RNAi substrate uptake to particular target cells [1][2][3]. More recently, researchers have been exploring the use of single-stranded toeholds and branch migration to induce formation of RNAi-activating RNA duplexes in a conditional fashion upon recognition of specified nucleic acid triggers [4][5][6]. Two new articles published by Bruce Shapiro and colleagues in Nano Letters explore the extent to which RNA toehold interactions can be used to prompt conditional RNAi-mediated gene silencing [7,8].…”

Activation of Multiple Functionalities Through Interacting Nanoparticles

“…To optimize the activity of nucleic acid nanoparticles, approaches to assembling dynamic RNA and RNA-DNA hybrid nanostructures have traditionally relied upon computer-assisted design and the use of single-stranded (ss) DNA or RNA toeholds for the resultant activation of switches or strand exchange [6][7][8][9][10] . We recently introduced the concept of interdependent, cognate nucleic acid nanoparticles that take advantage of shown to down-regulate apoptosis [15][16] , or against GFP were labeled with fluorophores chosen to undergo Förster resonance energy transfer (FRET).…”