Broccoli Fluorets: Split Aptamers as a User-Friendly Fluorescent Toolkit for Dynamic RNA Nanotechnology

Abstract

RNA aptamers selected to bind fluorophores and activate their fluorescence offer a simple and modular way to visualize native RNAs in cells. Split aptamers which are inactive until the halves are brought within close proximity can become useful for visualizing the dynamic actions of RNA assemblies and their interactions in real time with low background noise and eliminated necessity for covalently attached dyes. Here, we design and test several sets of F30 Broccoli aptamer splits, that we call fluorets, to compare their relative fluorescence and physicochemical stabilities. We show that the splits can be simply assembled either through one-pot thermal annealing or co-transcriptionally, thus allowing for direct tracking of transcription reactions via the fluorescent response. We suggest a set of rules that enable for the construction of responsive biomaterials that readily change their fluorescent behavior when various stimuli such as the presence of divalent ions, exposure to various nucleases, or changes in temperature are applied. We also show that the strand displacement approach can be used to program the controllable fluorescent responses in isothermal conditions. Overall, this work lays a foundation for the future development of dynamic systems for molecular computing which can be used to monitor real-time processes in cells and construct biocompatible logic gates.

Nucleic Acid Nanoparticles at a Crossroads of Vaccines and Immunotherapies

“…More importantly, NANPs technology is flexible and allows adding functionalities and swap individual nucleotides without altering the entire nanoparticle assembly [70]. As such, NANPs can be designed to form non-functional scaffolds, which are typically made of DNA, RNA or DNA-RNA hybrid oligonucleotides not-specific to any particular target gene, use these scaffolds to simultaneously deliver multiple different gene-specific therapeutic oligonucleotides (e.g., siRNAs or miRNAs) and create more sophisticated materials with conditionally activatable split functionalities [46,48,53,54,[65][66][67][68][70][71][72][73][74][75][76][77][78][79][80][81][82][83][84][85]. A co-delivery of individual non-functional scaffolds is required for applications involving NANPs with split functionalities and have been demonstrated in biological systems both in vitro and in vivo [53,73,74,77,82].…”

“…NANP's scaffolds were proven to deliver therapeutic oligonucleotides [66,72,77,121]. This modality may allow combining the beneficial immune response with additional therapeutic activities.…”

“…NANPs split functionality can be created when two individually non-functional NANPs are co-delivered into the same cell where they re-associate to create a fully functional NANP [76,77,123]. This modality provides both off and on switches to NANP-mediated biological responses.…”

“…When individual NANPs get off-target, the lack of their functionality at the individual level would prevent undesirable side effects. Afonin et al proposed an interesting concept in which an individual functional NANP is administered as a single therapeutic entity, then another NANP neutralizing the effect of the functional NANP is administered to quench the biological response and thereby avoid avert response [76,77,123]. This controlled "off" property is very attractive in immunotherapy, as it may provide control over the activated immune cells to avoid auto-immunity.…”

Co-transcriptional folding of a bio-orthogonal fluorescent scaffolded RNA origami

“…Since their development, fluorescent aptamers have been used in vitro and in vivo as a valid alternative to other imaging strategies (e.g. fluorescent in situ hybridization and molecular beacons, [55,56]). Malachite green, Mango, Spinach and Broccoli aptamers have been inserted in the design of different RNA nanostructures to successfully demonstrate the self-assembly and folding through their functional activation and fluorescence emission [19,24,29,53].…”