A Fluorescent Split Aptamer for Visualizing RNA-RNA AssemblyIn Vivo

Abstract

RNA-RNA assembly governs key biological processes and is a powerful tool for engineering synthetic genetic circuits. Characterizing RNA assembly in living cells often involves monitoring fluorescent reporter proteins, which are at best indirect measures of underlying RNA-RNA hybridization events and are subject to additional temporal and load constraints associated with translation and activation of reporter proteins. In contrast, RNA aptamers that sequester small molecule dyes and activate their fluorescence are increasingly utilized in genetically-encoded strategies to report on RNA-level events. Split-aptamer systems have been rationally designed to generate signal upon hybridization of two or more discrete RNA transcripts, but none directly function when expressed in vivo. We reasoned that the improved physiological properties of the Broccoli aptamer enable construction of a split-aptamer system that could function in living cells. Here we present the Split-Broccoli system, in which selfassembly is nucleated by a thermostable, three-way junction RNA architecture and fluorescence activation requires both strands. Functional assembly of the system approximately follows second order kinetics in vitro and improves when cotranscribed, rather than when assembled from purified components. Split-Broccoli fluorescence is digital in vivo and retains functional modularity when fused to RNAs that regulate circuit function through RNA-RNA hybridization, as demonstrated with an RNA Toehold switch. Split-Broccoli represents the first functional split-aptamer system to operate in vivo. It offers a genetically-encoded and nondestructive platform to monitor and exploit RNA-RNA hybridization, whether as an all-RNA, stand-alone AND gate or as a tool for monitoring assembly of RNA-RNA hybrids.

Rapid, Low-Cost Detection of Water Contaminants Using RegulatedIn VitroTranscription

“…To demonstrate the viability of an RNA output sensor, we chose the three-way junction dimeric Broccoli (3WJdB) aptamer [23], an engineered variant that scaffolds two monomers of Broccoli around a highly structured RNA three-way junction motif, and the fast phage RNA polymerase (RNAP) from T7 bacteriophage. T7 RNAP, unlike E. coli or other native host RNA polymerases, is a highly processive, single-subunit enzyme used in biotechnological applications that require high levels of target gene expression.…”

“…It is also extremely fast, with characterized transcription rates of 300 nucleotides (nt)/s in vitro [24]. To test the ability of T7 RNAP to drive 3WJdB synthesis in real-time, we PCR amplified a linear double-stranded DNA consisting of the T7 RNAP promoter followed by the 155 nt 3WJdB coding sequence and an optional 47 nt T7 transcription terminator [23] ( Fig. 2a).…”

TXO: Transcription-Only genetic circuits as a novel cell-free approach for Synthetic Biology

“…27 Since CFS are open and easily modifiable systems [11], time-consuming translation 28 and post-translational modification can be removed, allowing novel approaches with 29 faster signal generation. Alternative output signals independent of translation and 30 post-translational modification can provide the system with a simpler composition, 31 lower resource requirements and, most importantly, a shorter response time. Here, we 32 introduce a transcription-only (TXO) cell-free system using RNA aptamers as output 33 signals rather than translated proteins.…”

“…Further optimization should reduce these limits of detection. Previous works 212 with RNA aptamers for metal sensing, DasGupta et al mentioned the use of a truncated 213 form of the Spinach aptamer as a Pb 2+ sensor, but this could have lead to false results 214 at concentrations <2 ppm due to a lack of sensitivity and a weak output response [30]. 215 Since TXO systems are considerably simpler than TX-TL systems, with no require-216 ment for ribosomes, tRNA, or associated enzymes, we expect that it will be possible 217 to freeze-dry such systems for storage and distribution as previously reported for TX-218 TL systems [8,28].…”

“…using riboswitch aptamers) and signal processing 226 (e.g. using RNA-RNA hybridisation networks or crRNA/gRNA) [30][31][32][33][34][35].Possible im-227 provements to the already existing technology could also imply the use of other RNA 228 aptamers showing different properties, for instance, by improving the structure and 229 conditions of other aptamers published in the original work by Paige et al, which emit 230 8/16 fluorescence in different wavelength, i.e. red, yellow, cyan (see Table 1 and Figure 2) or 231 by using ribozymes, such as PS2.M, allowing additional possible transcriptional outputs 232 and broadening the possibilities of TXO systems.…”