Recurrent RNA motifs as scaffolds for genetically encodable small-molecule biosensors

Abstract

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.

Multi-state design of kinetically-controlled RNA aptamer ribosensors

“…The role of kinetic control mechanisms in creating biological activities that are both sensitive to, and selective for, specific molecular interactions has long been recognized 10 . Next-generation RNA aptamer biosensors have been successfully created through in vitro selection 40 , directed evolution 7 , activity-based screening 41,42 , two-state RNA folding design 8 , and thermodynamic optimization 43 . However, none of these state-of-the-art methods routinely generate RNA devices operating under kinetic control 7,8 , and an entirely different strategy was required to access that class of mechanisms.…”

“…4). Because the sensitivity of thermodynamically-controlled aptamer devices, including biosensors commonly engineered with the fluorogenic Spinach-family of aptamers, are fundamentally constrained by the equilibrium competition between the ON and OFF states 40,41,43 , they will always require concentrations significantly higher than the aptamer-ligand dissociation constant (K d ) for a response 13,36 . In this regard, it is very promising that the EC 50 s of the three kinetically-controlled p-AF ribosensors are both consistent with values predicted from the underlying design parameters ( Supplementary Fig.…”

RNA-based dynamic genetic controllers: development strategies and applications

“…Aptamers of natural riboswitches were utilized as scaffolds to select aptamers for new ligands. Nucleotides in the binding pockets of natural aptamers were randomized to produce potential aptamer libraries for in vitro selection (SELEX) [8 ] (Figure 1b). Selected aptamers for 5-hydroxytryptophan and 3,4-dihydroxyphenylalanine were successfully coupled to the readout domain, producing fluorescence upon binding of their cognate ligand in vitro and in vivo.…”

A mechanism for ligand gated strand displacement in ZTP riboswitch transcription regulation

“…The identification of riboswitch ligands has consistently revealed diverse roles for RNA molecules in cellular physiology and illustrated the capacity of RNA for precise chemical recognition (McCown, Corbino, Stav, Sherlock, & Breaker, 2017;Nelson & Breaker, 2017). In addition, riboswitches have found diverse utility as antibiotic targets (Howe et al, 2015), diagnostic biosensors (Kellenberger, Wilson, Sales-Lee, & Hammond, 2013;Porter, Polaski, Morck, & Batey, 2017), and as imaging tools (Braselmann et al, 2018).…”