Co-transcriptional production of RNA–DNA hybrids for simultaneous release of multiple split functionalities

Abstract

Control over the simultaneous delivery of different functionalities and their synchronized intracellular activation can greatly benefit the fields of RNA and DNA biomedical nanotechnologies and allow for the production of nanoparticles and various switching devices with controllable functions. We present a system of multiple split functionalities embedded in the cognate pairs of RNA–DNA hybrids which are programmed to recognize each other, re-associate and form a DNA duplex while also releasing the split RNA fragments which upon association regain their original functions. Simultaneous activation of three different functionalities (RNAi, Förster resonance energy transfer and RNA aptamer) confirmed by multiple in vitro and cell culture experiments prove the concept. To automate the design process, a novel computational tool that differentiates between the thermodynamic stabilities of RNA–RNA, RNA–DNA and DNA–DNA duplexes was developed. Moreover, here we demonstrate that besides being easily produced by annealing synthetic RNAs and DNAs, the individual hybrids carrying longer RNAs can be produced by RNA polymerase II-dependent transcription of single-stranded DNA templates.

Triggering RNAi with multifunctional RNA nanoparticles and their delivery

“…To introduce additional control over deliverable functionalities and to enchance their chemical stability, the properties of DNA and RNA were merged in the development of nanoparticles that were constructed from RNA/DNA hybrids [90,[96][97][98]. Combining the properties of these molecules (Figure 1, case 5 and Figure 2, right panel) allows for the splitting of the components of the functional elements (inactivating them) and permits their later activation under the control of complementary hybrids with ssDNA toeholds.…”

Computational and Experimental Studies of Reassociating RNA/DNA Hybrids Containing Split Functionalities

“…This concept has been expanded further to simultaneously release multiple split functionalities from two hybrid reassociations (Afonin, Desai, et al, 2014). As a proof of concept, we demonstrated the release of multiple split DS RNAs and RNA aptamers together with Förster resonance energy transfer (FRET) as shown in Fig.…”

“…Also, we were able to couple the hybrid concept with our multifunctional architectures such as nanocubes (Afonin et al, 2010, 2011; Afonin, Kasprzak, Bindewald, Kireeva, et al, 2014; Afonin, Kasprzak, Bindewald, Puppala, et al, 2014; Afonin, Viard, Kaglampakis, et al, In press). However, we demonstrated the use of RNA-based nanoparticles (nanorings; Afonin et al, 2011; Grabow et al, 2011; Yingling & Shapiro, 2007) that simultaneously activate hybrid split functions in cancer cells (Afonin, Desai, et al, 2014). Due to the increasing complexity of the hybrid structures, there is a great demand for computer algorithms that aim to assist in the design as well as the process of simulating the reassociation of the RNA/DNA hybrids.…”

Split Spinach Aptamer for Highly Selective Recognition of DNA and RNA at Ambient Temperatures

“…[8a] The probe was proven to be a versatile tool of RNA nanotechnology and synthetic biology. [9] The disadvantages of split MGA probe was low fluorescence intensity and strong photobleaching, which limited its practical applications. Spinach aptamer isolated by Paige et al [6] has attracted significant attention both as a tool for fluorescent monitoring of endogenous RNA in live cells [10] and as a sensor platform for detection of biological molecules and metal ions in vitro.…”