In vitro selected GUAA tetraloop-binding receptors with structural plasticity and evolvability towards natural RNA structural modules.

GNRA tetraloop-binding receptor interactions are key components in the macromolecular assembly of a variety of functional RNAs. In nature, there is an apparent bias for GAAA/11nt receptor and GYRA/helix interactions, with the former interaction being thermodynamically more stable than the latter. While past in vitro selections allowed isolation of novel GGAA and GUGA receptors, we report herein an in vitro selection that revealed several novel classes of specific GUAA receptors with binding affinities comparable to those from natural GAAA/11nt interactions. These GUAA receptors have structural homology with double-locked bulge RNA modules naturally occurring in ribosomal RNAs. They display mutational robustness that enables exploration of the sequence/phenotypic space associated to GNRA/receptor interactions through epistasis. Their thermodynamic self-assembly fitness landscape is characterized by a rugged neutral network with possible evolutionary trajectories toward natural GNRA/receptor interactions. High throughput sequencing analysis revealed synergetic mutations located away from the tertiary interactions that positively contribute to assembly fitness. Our study suggests that the repertoire of GNRA/receptor interactions is much larger than initially thought from the analysis of natural stable RNA molecules and also provides clues for their evolution towards natural GNRA/receptors.

Sensitive and Specific Cadmium Biosensor Developed by Reconfiguring Metal Transport and Leveraging Natural Gene Repositories.

Whole-cell biosensors are useful for monitoring heavy metal toxicity in public health and ecosystems, but their development has been hindered by intrinsic trade-offs between sensitivity and specificity. Here, we demonstrated an effective engineering solution by building a sensitive, specific, and high-response biosensor for carcinogenic cadmium ions. We genetically programmed the metal transport system of Escherichia coli to enrich intracellular cadmium ions and deprive interfering metal species. We then selected 16 cadmium-sensing transcription factors from the GenBank database and tested their reactivity to 14 metal ions in the engineered E. coli using the expression of the green fluorescent protein as the readout. The resulting cadmium biosensor was highly specific and showed a detection limit of 3 nM, a linear increase in fluorescent intensities from 0 to 200 nM, and a maximal 777-fold signal change. Using this whole-cell biosensor, a smartphone, and low-tech equipment, we developed a simple assay capable of measuring cadmium ions at the same concentration range in irrigation water and human urine. This method is user-friendly and cost-effective, making it affordable to screen large amounts of samples for cadmium toxicity in agriculture and medicine. Moreover, our work highlights natural gene repositories as a treasure chest for bioengineering.