Rapid and Multiplexed Nucleic Acid Detection using Programmable Aptamer-Based RNA Switches.

Rapid, simple, and low-cost diagnostic technologies are crucial tools for combatting infectious disease. Here, we describe a class of aptamer-based RNA switches called aptaswitches that recognize specific target nucleic acid molecules and respond by initiating folding of a reporter aptamer. Aptaswitches can detect virtually any sequence and provide a fast and intense fluorescent readout, generating signals in as little as 5 minutes and enabling detection by eye with minimal equipment. We demonstrate that aptaswitches can be used to regulate folding of six different fluorescent aptamer/fluorogen pairs, providing a general means of controlling aptamer activity and an array of different reporter colors for multiplexing. By coupling isothermal amplification reactions with aptaswitches, we reach sensitivities down to 1 RNA copy/µL in one-pot reactions. Application of multiplexed one-pot reactions against RNA extracted from clinical saliva samples yields an overall accuracy of 96.67% for detection of SARS-CoV-2 in 30 minutes. Aptaswitches are thus versatile tools for nucleic acid detection that can be readily integrated into rapid diagnostic assays.

Design of RNA-Based Translational Repressors.

The toehold switch is an RNA-based riboregulator that activates translation in response to a cognate trigger RNA and provides high ON/OFF ratios, excellent orthogonality, and logic capabilities. Riboregulators that provide the inverse function - turning off translation in response to a trigger RNA - are also versatile tools for sensing and efficiently implementing logic gates such as NAND or NOR. Toehold and three-way junction (3WJ) repressors are two de novo designed translational repressors devised to provide NOT functions with an easily programmable and intuitive structural design. Toehold and 3WJ repressors repress translation upon binding to cognate trigger RNAs by forming strong hairpin and three-way junction structures, respectively. These two translational repressors can be incorporated into multi-input NAND and NOR gates. This chapter provides methods for designing these translational repressors and protocols for in vivo characterization in E. coli.

Design of Ribocomputing Devices for Complex Cellular Logic.

The ability to control cell function is a critical goal for synthetic biology and motivates the development of ever-improving methods for precise regulation of gene expression. RNA-based systems represent powerful tools for this purpose since they can take full advantage of the predictable and programmable base pairing properties of RNA to control gene expression. This chapter is focused on the computational design of RNA-only biological circuits that can execute complex Boolean logic expressions in living cells. These ribocomputing devices use toehold switches as building blocks for circuit construction, integrating sensing, computation, and signal generation functions within a gate RNA transcript that regulates expression of a gene of interest. The gate RNA in turn assesses the assembly state of networks of interacting input RNAs to execute AND, OR, and NOT operations with high dynamic range in E. coli. Harnessing in silico tools for device design facilitates scaling of the circuits to complex logic expressions, including four-input AND, six-input OR, and disjunctive normal form expressions with up to 12 inputs. This molecular architecture provides an intuitive and modular strategy for devising logic systems that can be readily engineered using RNA sequence design software and applied in vivo and in vitro. In this chapter, we describe the process for designing ribocomputing devices from the generation of orthogonal toehold switch libraries through to their use as building blocks for AND, OR, and NOT circuitry.

Multi-arm RNA junctions encoding molecular logic unconstrained by input sequence for versatile cell-free diagnostics.

Applications of RNA-based molecular logic have been hampered by sequence constraints imposed on the input and output of the circuits. Here we show that the sequence constraints can be substantially reduced by appropriately encoded multi-arm junctions of single-stranded RNA structures. To conditionally activate RNA translation, we integrated multi-arm junctions, self-assembled upstream of a regulated gene and designed to unfold sequentially in response to different RNA inputs, with motifs of loop-initiated RNA activators that function independently of the sequence of the input RNAs and that reduce interference with the output gene. We used the integrated RNA system and sequence-independent input RNAs to execute two-input and three-input OR and AND logic in Escherichia coli, and designed paper-based cell-free colourimetric assays that accurately identified two human immunodeficiency virus (HIV) subtypes (by executing OR logic) in amplified synthetic HIV RNA as well as severe acute respiratory syndrome coronavirus-2 (via two-input AND logic) in amplified RNA from saliva samples. The sequence-independent molecular logic enabled by the integration of multi-arm junction RNAs with motifs for loop-initiated RNA activators may be broadly applicable in biotechnology.

De novo-designed translation-repressing riboregulators for multi-input cellular logic.

Efforts to construct synthetic biological circuits with more complex functions have often been hindered by the idiosyncratic behavior, limited dynamic range and crosstalk of commonly utilized parts. Here, we employ de novo RNA design to develop two high-performance translational repressors with sensing and logic capabilities. These synthetic riboregulators, termed toehold repressors and three-way junction (3WJ) repressors, detect transcripts with nearly arbitrary sequences, repress gene expression by up to 300-fold and yield orthogonal sets of up to 15 devices. Automated forward engineering is used to improve toehold repressor dynamic range and SHAPE-Seq is applied to confirm the designed switching mechanism of 3WJ repressors in living cells. We integrate the modular repressors into biological circuits that execute universal NAND and NOR logic and evaluate the four-input expression NOT ((A1 AND A2) OR (B1 AND B2)) in Escherichia coli. These capabilities make toehold and 3WJ repressors valuable new tools for biotechnological applications.

Self-assembly of DNA-oligo(p-phenylene-ethynylene) hybrid amphiphiles into surface-engineered vesicles with enhanced emission.

Surface-addressable nanostructures of linearly π-conjugated molecules play a crucial role in the emerging field of nanoelectronics. Herein, by using DNA as the hydrophilic segment, we demonstrate a solid-phase "click" chemistry approach for the synthesis of a series of DNA-chromophore hybrid amphiphiles and report their reversible self-assembly into surface-engineered vesicles with enhanced emission. DNA-directed surface addressability of the vesicles was demonstrated through the integration of gold nanoparticles onto the surface of the vesicles by sequence-specific DNA hybridization. This system could be converted to a supramolecular light-harvesting antenna by integrating suitable FRET acceptors onto the surface of the nanostructures. The general nature of the synthesis, surface addressability, and biocompatibility of the resulting nanostructures offer great promises for nanoelectronics, energy, and biomedical applications.

Fluorescence 'turn-on' sensor for F- derived from vitamin B6 cofactor.

A novel vitamin B6 Schiff base analog (L) was synthesized by combining vitamin B6 cofactor pyridoxal with 2-aminophenol. Receptor L displays a color change detectable by the naked-eye from yellow to red in the presence of fluoride and acetate due to the formation of hydrogen bonding host-guest complexes in 1 : 1 stoichiometry. Importantly, receptor L showed fluoride-selective 'turn-on' fluorescent response with a detection limit (3σ) of 7.39 × 10(-8) M.