Gene editing with CRISPR-Cas12a guides possessing ribose-modified pseudoknot handles

CRISPR-Cas12a is a leading technology for development of model organisms, therapeutics, and diagnostics. These applications could benefit from chemical modifications that stabilize or tune enzyme properties. Here we chemically modify ribonucleotides of the AsCas12a CRISPR RNA 5′ handle, a pseudoknot structure that mediates binding to Cas12a. Gene editing in human cells required retention of several native RNA residues corresponding to predicted 2′-hydroxyl contacts. Replacing these RNA residues with a variety of ribose-modified nucleotides revealed 2′-hydroxyl sensitivity. Modified 5′ pseudoknots with as little as six out of nineteen RNA residues, with phosphorothioate linkages at remaining RNA positions, yielded heavily modified pseudoknots with robust cell-based editing. High trans activity was usually preserved with cis activity. We show that the 5′ pseudoknot can tolerate near complete modification when design is guided by structural and chemical compatibility. Rules for modification of the 5′ pseudoknot should accelerate therapeutic development and be valuable for CRISPR-Cas12a diagnostics.

Impacts of Fluorescent Base Analogue Substitution on the Folding of a Riboswitch

Riboswitches are gene-regulating mRNA segments most commonly found in bacteria. A riboswitch contains an aptamer domain that binds to a ligand, causing a conformational change in a downstream expression platform. The aptamer domain of the Class I preQ1 riboswitch from Bacillus subtilis, which consists of a stem-loop structure and an adenine-rich single-stranded tail (L3), re-folds into a pseudoknot structure upon binding of its ligand, preQ1. To study the role of L3 in ligand recognition, we inserted 2-aminopurine (2-AP), a fluorescent base analogue of adenine (A), into the riboswitch at six different positions within L3. 2-AP differs from A in the relocation of its amino group from C6 to C2, allowing us to directly probe the significance of this specific functional group. We used circular dichroism spectroscopy and thermal denaturation experiments to study the structure and stability, respectively, of the riboswitch in the absence and presence of preQ1. At all labeling positions tested, 2-AP substitution inhibited the ability of preQ1 to stabilize the pseudoknot structure, with its location impacting the severity of the effect. Structural studies of the riboswitch suggest that at the most detrimental labeling sites, 2-AP substitution disrupts non-canonical base pairs. Our results show that these base pairs and tertiary interactions involving other residues in L3 play a critical role in ligand recognition by the preQ1 riboswitch, even at positions that are distal to the ligand binding pocket. They also highlight the importance of accounting for perturbations that fluorescent analogues like 2-AP may exert on the system being studied.

A combinatorial method to isolate short ribozymes from complex ribozyme libraries

In vitro selections are the only known methods to generate catalytic RNAs (ribozymes) that do not exist in nature. Such new ribozymes are used as biochemical tools, or to address questions on early stages of life. In both cases, it is helpful to identify the shortest possible ribozymes since they are easier to deploy as a tool, and because they are more likely to have emerged in a prebiotic environment. One of our previous selection experiments led to a library containing hundreds of different ribozyme clusters that catalyze the triphosphorylation of their 5′-terminus. This selection showed that RNA systems can use the prebiotically plausible molecule cyclic trimetaphosphate as an energy source. From this selected ribozyme library, the shortest ribozyme that was previously identified had a length of 67 nucleotides. Here we describe a combinatorial method to identify short ribozymes from libraries containing many ribozymes. Using this protocol on the library of triphosphorylation ribozymes, we identified a 17-nucleotide sequence motif embedded in a 44-nucleotide pseudoknot structure. The described combinatorial approach can be used to analyze libraries obtained by different in vitro selection experiments.

The conformational landscape of transcription intermediates involved in the regulation of the ZMP-sensing riboswitch from Thermosinus carboxydivorans

Recently, prokaryotic riboswitches have been identified that regulate transcription in response to change of the concentration of secondary messengers. The ZMP (5-Aminoimidazole-4-carboxamide ribonucleotide (AICAR))-sensing riboswitch from Thermosinus carboxydivorans is a transcriptional ON-switch that is involved in purine and carbon-1 metabolic cycles. Its aptamer domain includes the pfl motif, which features a pseudoknot, impeding rho-independent terminator formation upon stabilization by ZMP interaction. We herein investigate the conformational landscape of transcriptional intermediates including the expression platform of this riboswitch and characterize the formation and unfolding of the important pseudoknot structure in the context of increasing length of RNA transcripts. NMR spectroscopic data show that even surprisingly short pre-terminator stems are able to disrupt ligand binding and thus metabolite sensing. We further show that the pseudoknot structure, a prerequisite for ligand binding, is preformed in transcription intermediates up to a certain length. Our results describe the conformational changes of 13 transcription intermediates of increasing length to delineate the change in structure as mRNA is elongated during transcription. We thus determine the length of the key transcription intermediate to which addition of a single nucleotide leads to a drastic drop in ZMP affinity.

A functional RNA structure in the influenza A virus ribonucleoprotein complex for segment bundling

The influenza A virus genome is segmented into eight viral RNAs (vRNA). Intersegment interactions are necessary for segment bundling, and secondary structures on vRNA are assumed to be involved in the process. However, the RNA structure required for segment bundling remains unidentified because the secondary structure of vRNA in virion was partially unwound by binding viral non-specific RNA binding proteins. Here, we revealed the global intersegment interactions and the secondary structure of the vRNA in virion. We demonstrated that a pseudoknot structure was formed on a segment in the virion and the impairment of replication and packaging of the other specific segment was observed in cells infected with recombinant virus which had mutations in the pseudoknot structure. Moreover, we showed that the intersegment interactions were reconstituted in the recombinant virus. Our data provides the first evidence that the functional RNA structure on the influenza A virus genome affects segment bundling.

Evolving state grammar for modeling DNA and RNA structures

In this paper, we represent bio-molecular structures (Attenuator, Extended Pseudoknot Structure, Kissing Hairpin, Simple H-type structure, Recursive Pseudoknot and Three-knot Structure) using state grammar. These representations will be measured using descriptional complexity point of views. Results indicate that the proposed approach is more succinct in terms of production rules and variables over the existing approaches. Another major advantage of the proposed approach is state grammar can be represented by deep pushdown automata, whereas no such automaton exists for matrix ins-del system.