Novel approach using DNA-RNA hybrids in RNA nanotechnology

Re-association of RNA-DNA hybrids fluorescently labeled with dyes, Alexa488 and Alexa546, resulting in asymmetric Dicer substrate siRNA release.

Re-association of RNA-DNA hybrids fluorescently labeled with dyes, Alexa488 and Alexa546, resulting in asymmetric Dicer substrate siRNA release.

Developing simple approaches to detect interactions, modifications, and cellular locations of macromolecules is essential for understanding biochemical processes. The use of protein fragment complementation assays, also called split-protein systems, is a highly sensitive approach for studying protein interactions in biological systems. In this approach, functional proteins are split into non-functional fragments, and when attached to possible interacting partners, can reassemble and become functional again. Use of split-protein assays can establish differences between a healthy and a diseased state in the cell as well as determine the outcome of a therapeutic intervention.

In a proof-of-concept study, Bruce A. Shapiro, Ph.D., and his colleagues in CCR’s Nanobiology Program located at the NCI campus in Frederick, Maryland, set out to develop an approach similar to the split-protein system, but using nucleic acids rather than proteins. Since the discovery of RNA interference (RNAi), there has been great interest in using this technology for biomedical applications. RNAi refers to the posttranscriptional mechanism of potent and specific gene-silencing caused by double-stranded RNA (dsRNA) and a set of specific proteins and enzymes. Briefly, the RNaseIII-like enzyme, Dicer, cleaves dsRNA into short double-stranded RNA fragments called small interfering RNA (siRNA). The siRNAs are then loaded into a RNA-induced silencing complex (RISC), and one of the strands, called sense, is removed. The other strand, called antisense, is used by RISC to recognize the target mRNA for silencing. RNAi has potential to be useful in cancer and HIV therapeutics.

Previous studies have shown that while Dicer is able to cleave dsRNA, it cannot cleave RNA/DNA (R/DNA) hybrids. Also, substituting one or both siRNA strands with DNA inactivates RNAi. In an attempt to develop an approach that would allow for additional control over RNAi activation, and also potentially overcome some of the problems associated with stability and delivery of siRNAs, Shapiro’s team proposed to first split the functionality of Dicer substrate siRNAs into two R/DNA hybrids. Splitting dicer substrate siRNAs between two R/DNA hybrids prevents them from being diced and deemed non-functional. Then, self-recognizing complementary toeholds were added to each of the hybrid DNA strands. This results in rehybridization and release of Dicer substrate siRNA.

The researchers demonstrated the functionality of imaging the triggered response to delivery and re-association of hybrids in cells by fluorescently labeling the sense and antisense binding DNAs with dyes and performing Förster resonance energy transfer (FRET) studies. To determine if the hybrids could enter and re-associate within cells, the researchers co-transfected cells with hybrids fluorescently labeled with dyes and performed imaging using a confocal microscope. They observed that the fluorescent hybrids localized in endosomes of the cell. Additionally, in vitro experiments showed that hybrids were able to release functionalities, such as siRNAs, in cells.

In order to evaluate the delivery of hybrids in vivo, biodistribution experiments were done in athymic nude mice with xenograft tumors. Hybrids and siRNAs were first fluorescently labeled with a dye and then were administered to mice. Using fluorescent imaging, the researchers found that there was significant uptake of hybrids and siRNA by the tumor compared to other organs. Interestingly, the concentration of hybrids in mouse blood after three hours following the injection was almost two times higher than the concentration of siRNAs, which could indicate an increased stability of hybrids in blood compared to siRNA. Notably, the hybrid approach allowed for release of siRNAs against target genes in HIV and lung and breast cancer, with a prolonged effect of gene-silencing compared to the standard siRNA approach in vivo.

This work demonstrates a new approach to design and engineer R/DNA hybrid nanoparticles that can be used to trigger multiple functionalities when they re-associate inside cells. This novel strategy allows triggered release of therapeutic siRNAs inside diseased cells, activation of other split functionalities intracellularly such as FRET, and intracellular tracking of the delivery and re-association in real time. In regards to RNAi activation, the hybrids approach allows the triggered release of siRNAs in cell culture and in vivo with a longer effect of gene silencing compared to the siRNA in vivo. The approach may also permit a higher control over targeting specificity.

Importantly, the hybrid approach paves the way for further developments in the use of nucleic acid-based nanoparticles and switching devices conditionally releasing different functionalities for many nanotechnological and biomedical applications.

Summary Posted: Mon, 04/01/2013


Afonin KA, Viard M, Martins AN, Lockett SJ, Maciag AE, Freed EO, Heldman E, Jaeger L, Blumenthal R, Shapiro BA. Activation of different split functionalities on re-association of RNA-DNA hybrids. Nat Nanotechnol. 2013 Mar 31. PubMed Link