Systematic Mutation and Unnatural Base Pair Incorporation Improves Riboswitch-Based Biosensor Response Time.

Engineered RNAs have applications in diverse fields from biomedical to environmental. In many cases, the folding of the RNA is critical to its function. Here we describe a strategy to improve the response time of a riboswitch-based fluorescent biosensor. Systematic mutagenesis was performed to either make transpose or transition base pair mutants or introduce orthogonal base pairs. Both natural and unnatural base pair mutants were found to improve the biosensor response time without compromising fold turn-on or ligand affinity. These strategies can be transferred to improve the performance of other RNA-based tools.

Incorporation and Utility of a Responsive Ribonucleoside Analogue in Probing the Conformation of a Viral RNA Motif by Fluorescence and 19 F NMR Spectroscopy.

Development of versatile probes that can enable the study of different conformations and recognition properties of therapeutic nucleic acid motifs by complementing biophysical techniques can greatly aid nucleic acid analysis and therapy. Here, we report the design, synthesis and incorporation of an environment-sensitive ribonucleoside analogue, which serves as a two-channel biophysical platform to investigate RNA structure and recognition by fluorescence and 19 F NMR spectroscopy techniques. The nucleoside analogue is based on a 5-fluorobenzofuran-uracil core and its fluorescence and 19 F NMR chemical shifts are highly sensitive to changes in solvent polarity and viscosity. Notably, the modified ribonucleotide and phosphoramidite substrates can be efficiently incorporated into RNA oligonucleotides (ONs) by in vitro transcription and standard solid-phase ON synthesis protocol, respectively. Fluorescence and 19 F readouts of the nucleoside incorporated into model RNA ONs are sensitive to the neighbouring base environment. The responsiveness of the probe was aptly utilized in detecting and quantifying the metal ion-induced conformational change in an internal ribosome entry site RNA motif of hepatitis C virus, which is an important therapeutic target. Taken together, our probe is a good addition to the nucleic acid analysis toolbox with the advantage that it can be used to study nucleic acid conformation and recognition simultaneously by two biophysical techniques.

Live Cell Imaging Using Riboswitch-Spinach tRNA Fusions as Metabolite-Sensing Fluorescent Biosensors.

The development of fluorescent biosensors is motivated by the desire to monitor cellular metabolite levels in real time. Most genetically encodable fluorescent biosensors are based on receptor proteins fused to fluorescent protein domains. More recently, small molecule-binding riboswitches have been adapted for use as fluorescent biosensors through fusion to the in vitro selected Spinach aptamer, which binds a profluorescent, cell-permeable small molecule mimic of the GFP chromophore, DFHBI. Here we describe methods to prepare and analyze riboswitch-Spinach tRNA fusions for ligand-dependent activation of fluorescence in vivo. Example procedures describe the use of the Vc2-Spinach tRNA biosensor to monitor perturbations in cellular levels of cyclic di-GMP using either fluorescence microscopy or flow cytometry. In this updated chapter, we have added procedures on using biosensors in flow cytometry to detect exogenously added compounds. The relative ease of cloning and imaging of these biosensors, as well as their modular nature, should make this method appealing to other researchers interested in utilizing riboswitch-based biosensors for metabolite sensing.

Guanidine Biosensors Enable Comparison of Cellular Turn-on Kinetics of Riboswitch-Based Biosensor and Reporter.

Cell-based sensors are useful for many synthetic biology applications, including regulatory circuits, metabolic engineering, and diagnostics. While considerable research efforts have been made toward recognizing new target ligands and increasing sensitivity, the analysis and optimization of turn-on kinetics is often neglected. For example, to our knowledge there has been no systematic study that compared the performance of a riboswitch-based biosensor versus reporter for the same ligand. In this study, we show the development of RNA-based fluorescent (RBF) biosensors for guanidine, a common chaotropic agent that is a precursor to both fertilizer and explosive compounds. Guanidine is cell permeable and nontoxic to E. coli at millimolar concentrations, which in contrast to prior studies enabled direct activation of the riboswitch-based biosensor and corresponding reporter with ligand addition to cells. Our results reveal that the biosensors activate fluorescence in the cell within 4 min of guanidine treatment, which is at least 15 times faster than a reporter derived from the same riboswitch, and this rapid sensing activity is maintained for up to 1.6 weeks. Together, this study describes the design of two new biosensor topologies and showcases the advantages of RBF biosensors for monitoring dynamic processes in cell biology, biotechnology, and synthetic biology.

Synthesis and Enzymatic Incorporation of a Responsive Ribonucleoside Probe That Enables Quantitative Detection of Metallo-Base Pairs.

Synthesis of a highly responsive fluorescent ribonucleoside analogue based on a 5-methoxybenzofuran uracil core, enzymatic incorporation of its triphosphate substrate into RNA transcripts, and its utility in the specific detection and estimation of Hg2+-ion-mediated metallo-base pair formation in DNA-RNA and RNA-RNA duplexes are described.

Fluorescence-based tools to probe G-quadruplexes in cell-free and cellular environments.

Biophysical and biochemical investigations provide compelling evidence connecting the four-stranded G-quadruplex (GQ) structure with its role in regulating multiple cellular processes. Hence, modulating the function of GQs by using small molecule binders is being actively pursued as a strategy to develop new chemotherapeutic agents. However, sequence diversity and structural polymorphism of GQs have posed immense challenges in terms of understanding what conformation a G-rich sequence adopts inside the cell and how to specifically target a GQ motif amidst several other GQ-forming sequences. In this context, here we review recent developments in the applications of biophysical tools that use fluorescence readout to probe the GQ structure and recognition in cell-free and cellular environments. First, we provide a detailed discussion on the utility of covalently labeled environment-sensitive fluorescent nucleoside analogs in assessing the subtle difference in GQ structures and their ligand binding abilities. Furthermore, a detailed discussion on structure-specific antibodies and small molecule probes used to visualize and confirm the existence of DNA and RNA GQs in cells is provided. We also highlight the open challenges in the study of tetraplexes (GQ and i-motif structures) and how addressing these challenges by developing new tools and techniques will have a profound impact on tetraplex-directed therapeutic strategies.

A Dual-App Nucleoside Probe Provides Structural Insights into the Human Telomeric Overhang in Live Cells.

Understanding the topology adopted by individual G-quadruplex (GQ)-forming sequences in vivo and targeting a specific GQ motif among others in the genome will have a profound impact on GQ-directed therapeutic strategies. However, this remains a major challenge as most of the tools poorly distinguish different GQ conformations and are not suitable for both cell-free and in-cell analysis. Here, we describe an innovative probe design to investigate GQ conformations and recognition in both cell-free and native cellular environments by using a conformation-sensitive dual-app nucleoside analogue probe. The nucleoside probe, derived by conjugating fluorobenzofuran at the 5-position of 2'-deoxyuridine, is composed of a microenvironment-sensitive fluorophore and an in-cell NMR compatible 19F label. This noninvasive nucleoside, incorporated into the human telomeric DNA oligonucleotide repeat, serves as a common probe to distinguish different GQ topologies and quantify topology-specific binding of ligands by fluorescence and NMR techniques. Importantly, unique signatures displayed by the 19F-labeled nucleoside for different GQs enabled a systematic study in Xenopus laevis oocytes to provide new structural insights into the GQ topologies adopted by human telomeric overhang in cells, which so far has remained unclear. Studies using synthetic cell models, immunostaining on fixed cells, and crystallization conditions suggest that parallel GQ is the preferred conformation of telomeric DNA repeat. However, our findings using the dual-app probe clearly indicate that multiple structures including hybrid-type parallel-antiparallel and parallel GQs are formed in the cellular environment. Taken together, our findings open new experimental strategies to investigate topology, recognition, and therapeutic potential of individual GQ-forming motifs in a biologically relevant context.

Probing Human Telomeric DNA and RNA Topology and Ligand Binding in a Cellular Model by Using Responsive Fluorescent Nucleoside Probes.

The development of biophysical systems that enable an understanding of the structure and ligand-binding properties of G-quadruplex (GQ)-forming nucleic acid sequences in cells or models that mimic the cellular environment would be highly beneficial in advancing GQ-directed therapeutic strategies. Herein, the establishment of a biophysical platform to investigate the structure and recognition properties of human telomeric (H-Telo) DNA and RNA repeats in a cell-like confined environment by using conformation-sensitive fluorescent nucleoside probes and a widely used cellular model, bis(2-ethylhexyl) sodium sulfosuccinate reverse micelles (RMs), is described. The 2'-deoxy and ribonucleoside probes, composed of a 5-benzofuran uracil base analogue, faithfully report the aqueous micellar core through changes in their fluorescence properties. The nucleoside probes incorporated into different loops of H-Telo DNA and RNA oligonucleotide repeats are minimally perturbing and photophysically signal the formation of respective GQ structures in both aqueous buffer and RMs. Furthermore, these sensors enable a direct comparison of the binding affinity of a ligand to H-Telo DNA and RNA GQ structures in the bulk and confined environment of RMs. These results demonstrate that this combination of a GQ nucleoside probe and easy-to-handle RMs could provide new opportunities to study and devise screening-compatible assays in a cell-like environment to discover GQ binders of clinical potential.