Abstract
RNA aptamers that bind non-fluorescent dyes and activate their fluorescence are highly sensitive, nonperturbing, and convenient probes in the field of synthetic biology. These RNA molecules, referred to as light-up aptamers, operate as molecular nanoswitches that alter folding and fluorescence function in response to ligand binding, which is important in biosensing and molecular computing. Herein, we demonstrate a conceptually new generation of smart RNA nano-devices based on malachite green (MG)-binding RNA aptamer, which fluorescence output controlled by addition of short DNA oligonucleotides inputs. Four types of RNA switches possessing AND, OR, NAND, and NOR Boolean logic functions were created in modular form, allowing MG dye binding affinity to be changed by altering 3D conformation of the RNA aptamer. It is essential to develop higher-level logic circuits for the production of multi-task nanodevices for data processing, typically requiring combinatorial logic gates. Therefore, we further designed and synthetized higher-level half adder logic circuit by “in parallel” integration of two logic gates XOR and AND within a single RNA nanoparticle. The design utilizes fluorescence emissions from two different RNA aptamers: MG-binding RNA aptamer (AND gate) and Broccoli RNA aptamer that binds DFHBI dye (XOR gate). All computationally designed RNA devices were synthesized and experimentally tested in vitro. The ability to design smart nanodevices based on RNA binding aptamers offers a new route to engineer “label-free” ligand-sensing regulatory circuits, nucleic acid detection systems, and gene control elements.
Highlights
The progression in the field of RNA nanotechnology makes RNA molecules the most promising candidate to fabricate bio-computers due to their variable folding properties as well as their catalytic functions [1,2]
The structures of four logic gates were designed based on the general 2D architecture depicted in Figure 2 and all functioned as robust RNA switches that exhibit fluorescence emission once activated
The design process used here accounts for the thermodynamic stability of various base-paired structures in the absence or presence of input oligonucleotides
Summary
The progression in the field of RNA nanotechnology makes RNA molecules the most promising candidate to fabricate bio-computers due to their variable folding properties as well as their catalytic functions [1,2]. TThhee rruulleess ssppeecciiffiieeddbbyyeeaacchhggaatteeaarreesshhoowwnninintrturuththtatabblelsestotoththee lelefftt..NNoorrmmaalliizzeedd flfluuoorreesscceennccee eennhhaanncceemmeenntt ooff tthhee ggaatteessiissddiissppllaayyeeddiinntthheemmididddlele. The AND and OR gates with default setting (0-0; no inputs present) was designed using interfering ends The ssDNA inputs were designed to have several extra nucleotides to achieve an equilibrium state where inclusion of at least one input will trigger conformational change in favor of the correctly folded MG-binding RNA aptamer. The DNA inputs were selected to contain longer sequences with much higher Tm values as compared to the AND gate, so that disrupted hairpin nts initiate a conformational change of the whole construct favoring formation of the ON aptamer state (Figure S2, OR GATE). The extent to which these modular RNA logic gates can be used to probe three or more inputs simultaneously will depend on their reliability in tandem
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