RNA a-site Identification via Multi-Type Nucleic Acid Screening
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Solution Overview
Problem
Current methods for detecting accessible regions (a-sites) in complex RNA molecules are inefficient, leading to limited silencing efficacy due to the structural complexity of RNA molecules, which hinders the binding of directing nucleic acids and endonucleases, resulting in incomplete target specificity and off-target effects.
Innovation Solution
A method involving an esiRNA/ERNA screen to identify siRNAs that bind effectively to AGO proteins, followed by assays with antisense DNA oligonucleotides and g/crRNAs to determine functional changes in target RNAs, identifying a-sites accessible to various nucleic acid-endonuclease complexes for precise modification.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If current methods are used to detect accessible regions in complex RNA molecules, then the process is simpler, but the detection precision and reliability are insufficient due to RNA structural complexity
Solution Approach 1:
The method segments the detection process into multiple distinct steps: (i) esiRNA/ERNA screening to identify functional siRNAs, (ii) validation with antisense DNA oligonucleotides, and (III) verification with g/crRNAs. Each step narrows down the identification of true a-sites, progressively improving detection precision while managing overall method complexity through systematic division of the detection task.
Solution Approach 2:
The method employs multiple types of directing nucleic acids (esiRNAs/ERNAs, antisense DNA oligonucleotides, g/crRNAs) that can all bind to the same a-sites but through different mechanisms. This multi-functional approach allows verification of a-site accessibility from multiple angles, significantly improving detection reliability and precision by cross-validating results across different nucleic acid types.
2Reliability
If directing nucleic acids are used to target RNA molecules, then gene expression can be modulated, but off-target effects occur due to incomplete target specificity
Solution Approach 1:
The method performs preliminary identification and validation of a-sites using multiple directing nucleic acid types before final targeting. By pre-screening with esiRNAs/ERNAs, validating with antisense DNA oligonucleotides, and verifying with g/crRNAs, the method ensures that only truly accessible and specific sites are targeted, preventing off-target effects before they occur.
Solution Approach 2:
The multi-step method incorporates feedback mechanisms where each validation step informs the next. Results from esiRNA/ERNA screening feed into antisense oligonucleotide validation, which in turn informs g/crRNA verification. This feedback loop continuously refines target specificity and eliminates potential off-target sites, ensuring high reliability of the final targeting.
3Productivity
If RNA molecules are targeted for silencing, then gene expression can be controlled, but silencing efficacy is limited due to RNA structural complexity
Solution Approach 1:
The method changes the parameter of nucleic acid type used for targeting, transitioning from single-type to multi-type directing nucleic acids. By employing esiRNAs/ERNAs, antisense DNA oligonucleotides, and g/crRNAs with different chemical properties and binding characteristics, the method overcomes RNA structural barriers that limit single-type targeting, thereby improving silencing efficacy despite RNA complexity.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
This approach allows for reliable identification of a-sites, enhancing the efficacy of RNA-mediated, DNA-mediated, and CRISPR/Cas-mediated silencing by ensuring precise targeting and reducing off-target effects, thereby improving pathogen control and gene expression modulation.
Implementation Method 1
Different types of short-chain nucleic acids (NAs), also referred to as 'directing NAs' in the following, are associated with target RNAs via complementary base pairing and thus very specifically.
Implementation Method 2
The directing NAs can already inhibit the target RNA in its function, e.g. as a translation substrate, by binding ('hybridization') to it.
Implementation Method 3
subsequent assay with derived antisense DNA oligonucleotides (ASO) to determine whether they can induce a functional change in the target RNA in the presence or absence of RNase H
Implementation Method 4
subsequent testing with g/crRNAs derived therefrom to determine whether they can induce a functional change in the target RNA in the presence of a Cas protein
Implementation Method 5
The function of the target RNAs can then be inhibited via nuclease-mediated catalysis of an endonucleolytic cleavage.
Data Source
AI summary
The invention relates to a method for detecting accessible regions (‘a sites’) in complex RNA molecules (target RNAs), wherein nucleic acids or complexes of these nucleic acids and endonucleases associated therewith bind to the a-sites and alter the function of the target RNAs where by nucleic acids of different types (‘eNAs’) bind to the a-sites and either alone or through an associated endonuclease alter the function of this target RNA. The invention further relates to the use of the method for identifying nucleic acids of different types eNAs that bind to the a-sites and are capable of directing endonucleases, selected from AGO proteins, RNase H and Cas proteins to the a-sites of target RNAs and are capable of, in the presence or absence of these endonucleases reliably affecting/altering the function of these RNA molecules; and to eNAs and a composition containing these eNAs in pathogen control.