Programmable RNA detection with a fluorescent RNA aptamer using optimized three-way junction formation

Abstract

RNAs play essential roles in various cellular processes and can be used as biomarkers. Hence, it is important to detect endogenous RNA for understanding diverse cellular functions and diagnosing diseases. To construct a low-cost and easy-to-use RNA detection probe, a chemically unmodified RNA aptamer that binds to a pro-fluorophore to increase its fluorescence is desirable. Here, we focused on Broccoli, a superior variant of Spinach, which is a well-known fluorescent RNA aptamer that binds to DFHBI-1T and emits green fluorescence. We experimentally characterized Broccoli and predicted that it forms a G-quadruplex–based DFHBI-1T recognition region sandwiched between two stems. Based on this, we designed a Broccoli-based RNA detection probe (BRD probe) composed of a sequence of destabilized Broccoli fused with complementary sequences against target RNA. The resulting probe with its target RNA formed a stable three-way junction, named the MT2 three-way junction, which contributed to efficient refolding of the Broccoli structure and allowed for programmable RNA detection with high signal-to-noise ratio and sensitivity. Interestingly, the MT2 three-way junction also could be applied to probe construction of a truncated form of Spinach (Baby Spinach). The BRD and Baby Spinach–based RNA detection probes (BSRD probe) exhibited up to 48- and 140-fold fluorescence enhancements in the presence of their target RNAs and detected small amounts of target RNA that were as low as 160 and 5 nM, respectively. Thus, we experimentally characterized the higher order structure of Broccoli and developed structure-switching aptamer probes for highly sensitive, programmable, RNA detection using an MT2 three-way junction.

Co-transcriptional RNA strand displacement circuits

“…in blood samples (17), cell-free lysates, or inside living cells (18). In vivo, fluorescent RNA aptamers (46,47) or RNA regulators that transduce RNA signals into fluorescent protein production (24) could track ctRSD dynamics. Further, ctRSD outputs could regulate protein expression through existing RNA technologies (22,23,27), allowing ctRSD circuits to control cellular function.…”

“…2C). We opted to use a DNA-based reporter, rather than an RNA aptamerbased reporter (46,47), because the DNA reporter is easily calibrated to output concentration for modeling (4,5,7). To be stable at 37 o C, we designed the reporter with a 16 base duplex.…”

In Vivo Production of RNA Aptamers and Nanoparticles: Problems and Prospects

“…The Broccoli aptamer has a higher affinity for fluorophores and the Broccoli–DFHBI-1T complex displays a brighter signal than Spinach–DFHBI [ 84 ]. Broccoli retains most of the G-quadruplex-forming nucleotides from the DFHBI-binding pocket in Spinach2 and probably has a similar structure upon interaction with DFHBI-1T [ 86 , 96 , 97 ] ( Figure 2 ).…”

Development and Applications of Fluorogen/Light-Up RNA Aptamer Pairs for RNA Detection and More

“…In addition to the Spinach family aptamers, it is very likely that the same DFHBI(-1T)-binding pocket is shared by Broccoli [35]. Indeed, even though no crystal structure has been solved for this aptamer, the close sequence proximity with Spinach strongly suggests that both molecules adopt the same folding [59][60][61]. However, despite the tight apparent accommodation of the fluorogen, all the known DFHBI(-1T)/aptamer complexes suffer from a very short fluorescence half-life (less than 1 second) [23].…”

“…Such approach was successfully applied to endogenous mRNA visualization using engineered versions of the BHQ-1 binding aptamer [72] and of Spinach [73]. Recently, improved designs of Broccoli-derived probes with optimized sequences have been proposed and were shown to display superior performances to detect mRNA in vitro [61,74]. Yet, these probes still need to be validated in live cells.…”