Structures of riboswitch receptor domains bound to their effector have revealed how mRNAs recognize diverse small molecules, but mechanistic details into its linkage with regulation of gene expression remain elusive1,2. To address this, we solved crystal structures of two different classes of cobalamin (vitamin B12) binding riboswitches that include the structural switch of the downstream regulatory domain. These classes share a common cobalamin-binding core, but use distinct peripheral extensions to recognize different B12 derivatives. In each case, recognition is accomplished through shape complementarity between the RNA and cobalamin with relatively few hydrogen bonding interactions that typically govern RNA-small molecule recognition. We show that a composite cobalamin/RNA scaffold stabilizes an unusual long-range intramolecular kissing-loop interaction that controls mRNA expression. This is the first riboswitch crystal structure detailing how the receptor and regulatory domains communicate in a ligand-dependent fashion to regulate mRNA expression.

Allosteric RNA devices are increasingly viewed as important tools capable of monitoring enzyme evolution, optimizing engineered metabolic pathways, facilitating gene discovery and regulators of nucleic acid-based therapeutics. A key bottleneck in the development of these platforms is the availability of small molecule binding RNA aptamers that robustly function in the cellular environment. While aptamers can be raised against nearly any desired target by in vitro selection, many cannot be easily integrated into devices or do not reliably function in a cellular context. Here, we describe a new approach using secondary and tertiary structural scaffolds derived from biologically active riboswitches and small ribozymes. Applied to neurotransmitter precursors 5-hydroxytryptophan and 3,4-dihydroxyphenylalanine, this approach yields easily identifiable and characterizable aptamers predisposed for coupling to readout domains to engineer nucleic acid sensory devices that function in vitro and in the cellular context.

RNAs directly regulate a vast array of cellular processes, emphasizing the need for robust approaches to fluorescently label and track RNAs in living cells. Here, we develop an RNA imaging platform using the cobalamin riboswitch as an RNA tag and a series of probes containing cobalamin as a fluorescence quencher. This highly modular 'Riboglow' platform leverages different colored fluorescent dyes, linkers and riboswitch RNA tags to elicit fluorescence turn-on upon binding RNA. We demonstrate the ability of two different Riboglow probes to track mRNA and small noncoding RNA in live mammalian cells. A side-by-side comparison revealed that Riboglow outperformed the dye-binding aptamer Broccoli and performed on par with the gold standard RNA imaging system, the MS2-fluorescent protein system, while featuring a much smaller RNA tag. Together, the versatility of the Riboglow platform and ability to track diverse RNAs suggest broad applicability for a variety of imaging approaches.

Riboswitches are a widely distributed class of regulatory RNAs in bacteria that modulate gene expression via small-molecule-induced conformational changes. Generally, these RNA elements are grouped into classes based upon conserved primary and secondary structure and their cognate effector molecule. Although this approach has been very successful in identifying new riboswitch families and defining their distributions, small sequence differences between structurally related RNAs can alter their ligand selectivity and regulatory behavior. Herein, we use a structure-based mutagenic approach to demonstrate that cobalamin riboswitches have a broad spectrum of preference for the two biological forms of cobalamin using isothermal titration calorimetry. This selectivity is primarily mediated by the interaction between a peripheral element of the RNA that forms a T-loop module and a subset of nucleotides in the cobalamin-binding pocket. Cell-based fluorescence reporter assays in revealed that mutations that switch effector preference lead to differential regulatory responses in a biological context. These data demonstrate that a more comprehensive analysis of representative sequences of both previously and newly discovered classes of riboswitches might reveal subgroups of RNAs that respond to different effectors. Furthermore, this study demonstrates a second distinct means by which tertiary structural interactions in cobalamin riboswitches dictate ligand selectivity.