Toehold-Mediated Strand Displacement in Random Sequence Pools.

Toehold-mediated strand displacement (TMSD) has been used extensively for molecular sensing and computing in DNA-based molecular circuits. As these circuits grow in complexity, sequence similarity between components can lead to cross-talk, causing leak, altered kinetics, or even circuit failure. For small non-biological circuits, such unwanted interactions can be designed against. In environments containing a huge number of sequences, taking all possible interactions into account becomes infeasible. Therefore, a general understanding of the impact of sequence backgrounds on TMSD reactions is of great interest. Here, we investigate the impact of random DNA sequences on TMSD circuits. We begin by studying individual interfering strands and use the obtained data to build machine learning models that estimate kinetics. We then investigate the influence of pools of random strands and find that the kinetics are determined by only a small subpopulation of strongly interacting strands. Consequently, their behavior can be mimicked by a small collection of such strands. The equilibration of the circuit with the background sequences strongly influences this behavior, leading to up to 1 order of magnitude difference in reaction speed. Finally, we compare two established and one novel technique that speed up TMSD reactions in random sequence pools: a three-letter alphabet, protection of toeholds by intramolecular secondary structure, or by an additional blocking strand. While all of these techniques were useful, only the latter can be used without sequence constraints. We expect that our insights will be useful for the construction of TMSD circuits that are robust to molecular noise.

Algorithmic Design of 3D Wireframe RNA Polyhedra.

We address the problem of de novo design and synthesis of nucleic acid nanostructures, a challenge that has been considered in the area of DNA nanotechnology since the 1980s and more recently in the area of RNA nanotechnology. Toward this goal, we introduce a general algorithmic design process and software pipeline for rendering 3D wireframe polyhedral nanostructures in single-stranded RNA. To initiate the pipeline, the user creates a model of the desired polyhedron using standard 3D graphic design software. As its output, the pipeline produces an RNA nucleotide sequence whose corresponding RNA primary structure can be transcribed from a DNA template and folded in the laboratory. As case examples, we design and characterize experimentally three 3D RNA nanostructures: a tetrahedron, a triangular bipyramid, and a triangular prism. The design software is openly available and also provides an export of the targeted 3D structure into the oxDNA molecular dynamics simulator for easy simulation and visualization.

Controlling Gene Expression in Mammalian Cells Using Multiplexed Conditional Guide RNAs for Cas12a*.

Spatiotemporal control of the activity of CRISPR-associated (Cas) proteins is of considerable interest for basic research and therapeutics. Here, we show that conditional guide RNAs (gRNAs) for Cas12a can be transcribed in mammalian cells by RNA polymerase II, followed by activation via input-dependent processing of the 3' tail of the gRNA transcript. We demonstrate processing using an RNA strand displacement mechanism, as well as microRNA-dependent processing, and cleavage by a guanine-responsive ribozyme. We further demonstrate that Cas12a along with several independently switchable gRNAs can be compactly integrated on a single transcript using stabilizing RNA triplexes, providing a route towards Cas12a-based gene regulation constructs with multi-input switching capabilities. The principle is shown to work in HEK and mouse fibroblast cells using luminescence, fluorescence, and is also demonstrated for the conditional upregulation of an endogenous gene.

Switching the activity of Cas12a using guide RNA strand displacement circuits.

The CRISPR effector protein Cas12a has been used for a wide variety of applications such as in vivo gene editing and regulation or in vitro DNA sensing. Here, we add programmability to Cas12a-based DNA processing by combining it with strand displacement-based reaction circuits. We first establish a viable strategy for augmenting Cas12a guide RNAs (gRNAs) at their 5' end and then use such 5' extensions to construct strand displacement gRNAs (SD gRNAs) that can be activated by single-stranded RNA trigger molecules. These SD gRNAs are further engineered to exhibit a digital and orthogonal response to different trigger RNA inputs-including full length mRNAs-and to function as multi-input logic gates. We also demonstrate that SD gRNAs can be designed to work inside bacterial cells. Using such in vivo SD gRNAs and a DNase inactive version of Cas12a (dCas12a), we demonstrate logic gated transcriptional control of gene expression in E. coli.

In Situ Monitoring the Uptake of Moisture into Hybrid Perovskite Thin Films.

Solution-processed hybrid perovskites are of great interest for use in photovoltaics. However, polycrystalline perovskite thin films show strong degradation in humid atmospheres, which poses an important challenge for large-scale market introduction. With in situ grazing incidence neutron scattering (GISANS) we analyzed water content, degradation products, and morphological changes during prolonged exposure to several humidity levels. In high humidity, the formation of metastable hydrate phases is accompanied by domain swelling, which transforms the faceted crystals to a round-washed, pebble-like form. The films incorporate much more water than is integrated into the hydrates, with smaller crystals being more affected, making the degradation strongly dependent on film morphology. Even at low humidity, water is adsorbed on the crystal surfaces without the formation of crystalline degradation products. Thus, although production in an ambient atmosphere is of interest for industrial production it might lead to long-term degradation without appropriate countermeasures like postproduction drying below 30% RH.

Nanostructuring Mixed-Dimensional Perovskites: A Route Toward Tunable, Efficient Photovoltaics.

2D perovskites is one of the proposed strategies to enhance the moisture resistance, since the larger organic cations can act as a natural barrier. Nevertheless, 2D perovskites hinder the charge transport in certain directions, reducing the solar cell power conversion efficiency. A nanostructured mixed-dimensionality approach is presented to overcome the charge transport limitation, obtaining power conversion efficiencies over 9%.