RNA Promoter Identification via Ribozyme Cleavage and Rolling Circle Transcription
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Solution Overview
Problem
Current methods for identifying RNA promoters are inadequate as they do not yield cloned promoters or allow for promoter mutation evaluation, and existing technologies rely on bioinformatics analysis of viral genomes to find similar regions, which is inefficient.
Innovation Solution
The development of DNA and RNA constructs comprising specific operably linked polynucleotide elements, including ribozyme catalytic cores and cleavage sites, which are transfected into host cells to identify functional RNA promoters through rolling circle transcription and sequencing.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If bioinformatics analysis of viral genomes is used to identify similar regions, then promoter identification can be performed, but the method does not yield cloned promoters or allow promoter mutations to be evaluated
Solution Approach 1:
The method segments the promoter identification process into distinct functional components: a promoter region, a ribozyme element, and a reporter gene. This segmentation allows the promoter to be isolated and evaluated independently through functional assays, enabling both identification and mutation analysis while maintaining measurement precision.
Solution Approach 2:
The patent introduces a ribozyme element as an intermediary between the promoter and reporter gene. This intermediary serves as a molecular marker that enables cloning and functional evaluation of promoters, bridging the gap between bioinformatics identification and experimental validation while allowing promoter mutations to be assessed.
2Measurement precision
If individual research into viral genomic and subgenomic promoters is conducted, then specific promoter sequences can be identified, but the process is inefficient and does not yield cloned promoters
Solution Approach 1:
The patent creates a universal multi-functional construct that can simultaneously serve as a cloning vector, expression system, and evaluation platform. The standardized design with promoter, ribozyme, and reporter gene elements allows efficient high-throughput screening of multiple promoters, improving productivity while maintaining sequence identification precision.
Solution Approach 2:
The method enables systematic variation of promoter sequences through controlled mutagenesis while maintaining the overall construct architecture. By changing promoter parameters (sequence, length, mutations) within the standardized framework, researchers can efficiently evaluate multiple variants, significantly improving research productivity without sacrificing identification accuracy.
3Loss of information
If the sequence flanking the 5′end of an expressed subgenomic sequence is identified as the promoter, then promoter location can be determined, but the cloned promoter cannot be obtained or evaluated
Solution Approach 1:
The patent employs preliminary action by designing the construct with the promoter already positioned upstream of the ribozyme element before transfection. This pre-arranged configuration ensures that when the construct is introduced into cells, the promoter is immediately in the correct position to drive transcription, allowing both location determination and direct cloning of functional promoters without additional manipulation.
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 enables the identification and evaluation of RNA promoters by producing circularized RNA containing ribozyme cores and primer annealing sites, allowing for the isolation and sequencing of functional promoters, thereby overcoming the limitations of existing methods.
Implementation Method 1
a hammerhead ribozyme catalytic core; iv) a first hairpin ribozyme cleavage site in the antisense orientation; vii) a hairpin ribozyme catalytic core in the antisense orientation
Implementation Method 2
transcribing in vitro into RNA the DNA construct as described above and herein, thereby producing a RNA transcript of the DNA construct
Implementation Method 3
wherein the host cell expresses a RNA-dependent RNA polymerase and rolling circle transcription of the RNA construct or RNA transcribed from the DNA construct within the host cell when the inserted polynucleotide comprises a functional RNA promoter yields a circularized RNA
Implementation Method 4
amplifying the inserted polynucleotide comprising a functional RNA promoter; and d) sequencing the inserted polynucleotide comprising a functional RNA promoter
Data Source
AI summary
Provided are constructs and methods for RNA promoter identification.