Compositions and methods relating to nucleic acid interaction reporters
Inventors
CIFUENTES BUIRA, Daniel • KRETOV, Dmitry
Assignees
Publication Number
US-12258625-B2
Publication Date
2025-03-25
Expiration Date
2043-04-04
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Abstract
The technology described herein is directed to compositions, methods, and systems for measuring the interactions between RNAs and proteins.
Core Innovation
The invention provides compositions, methods, and systems for detecting and quantifying interactions between proteins with RNA-binding domains and their cognate RNA sequences. It introduces a paired approach utilizing a polypeptide or pair of polypeptides that include both a candidate RNA-binding domain and the catalytic domain of an RNA-editing enzyme, along with an RNA containing both a candidate binding site for the RNA-binding domain and a cognate substrate for the catalytic domain. This design allows reliable targeting and measurement of protein-RNA binding interactions.
A core problem addressed by this technology is the limitation of prior art methods, which depend on engineered proteins targeting endogenous mRNAs that are often unsuitable targets. Existing techniques are constrained by the lack of adequate substrate sites in natural RNAs and by binary or biased assay outputs. The disclosed technology overcomes these issues by providing a universal, high-sensitivity system enabling quantitative analysis of RNA-protein interactions without relying on endogenous mRNAs.
The system enables the study of not only the presence or half-life of mRNAs but directly assesses the interaction itself between RNA-binding proteins and their target RNAs under various conditions, including multiplexed formats. The strength of the interaction is registered via catalytic RNA editing events, which can be detected and quantified by sequencing, fluorescence, or reporter gene activity. This approach facilitates the systematic screening of interaction pairs, the effect of modifiers or compounds, and supports generation of datasets for understanding protein-RNA recognition.
Claims Coverage
The patent contains independent claims that define several main inventive features concerning compositions, methods, and cellular systems for measuring the interactions between RNA-binding proteins and RNA using catalytic RNA editing.
Combination of polypeptide(s) with RNA-binding domain and RNA-editing enzyme with RNA containing binding and substrate sites
A combination comprising: - At least one polypeptide or pair of polypeptides that includes (i) a candidate RNA-binding domain and (ii) a catalytic domain of an RNA-editing enzyme. - At least one RNA that has (i) at least one candidate cognate binding site for the candidate RNA-binding domain and (ii) a plurality of instances or occurrences of a cognate substrate site for the catalytic domain. - The candidate cognate binding site and the cognate substrate site are not both found in the same naturally-occurring RNA.
Cell comprising or expressing the protein-RNA combination
A cell comprising or expressing the combination described above, i.e., expressing one or more polypeptides comprising a candidate RNA-binding domain and a catalytic domain of an RNA-editing enzyme, together with at least one RNA containing a candidate cognate binding site and a cognate substrate site.
Method for detecting strength of binding using editing readout
A method comprising: 1. Contacting the at least one polypeptide as described, with the at least one RNA as described, for a period of time. 2. Detecting the amount of editing present in the cognate substrate site. The amount of editing correlates to the strength of the binding between the candidate RNA-binding domain and the candidate cognate binding site.
The inventive features include a defined combination of engineered polypeptides and RNAs for non-natural measurement of RNA-protein binding via RNA editing, methods for quantifying binding interaction strength through detectable editing, and cells expressing these system components.
Stated Advantages
The technology provides quantitative analysis of protein-RNA interactions, overcoming the binary outputs of prior art techniques such as TRIBE.
It is unbiased and avoids methodological biases present in cross-linking techniques like CLIP, which relies on covalent cross-linking with UV irradiation.
The system allows for universal study of protein-mRNA binding interactions with high sensitivity, without being restricted by endogenous mRNA suitability.
The approach enables in vivo analysis, capturing the nuances of protein-RNA interactions within living cells, making it closer to therapeutic scenarios.
It provides a broad dynamic range, with signal being responsive to both time and dosage of interacting components.
Documented Applications
Screening for small molecules (such as drugs, antibiotics, or herbicides) that modulate RNA-binding domain-cognate binding site interactions.
Identifying and quantifying binding motifs and preferred binding sites of RNA-binding proteins across transcriptomes and different cell types.
Studying the binding of readers, writers, or erasers of epitranscriptomic modifications, including proteins that recognize m6A methylation in RNA.
Engineering and screening artificial RNA-binding proteins, such as designer proteins based on PUF domain arrays, for binding to sequences of interest.
Monitoring microRNA activity in vivo, including measuring the interaction strength of microRNAs with their targets and identifying the most active microRNAs in cells.
Detecting protein-protein interactions using a system where interaction-dependent proximity leads to RNA editing as a readout.
Dissecting the structural determinants of RNA-protein recognition by testing different structured RNA elements for binding and resultant editing.
Measuring translation efficiency by fusing ribosomal proteins to the RNA-editing enzyme and coupling editing to ribosomal activity on a reporter mRNA.
Evaluating how post-translational modifications of RNA-binding proteins modulate RNA-binding activity, using mutant and wild-type forms and measuring editing outcomes.
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