Universal mRNA Enrichment via Poly(A) Polymerase and IFIT Binding
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Solution Overview
Problem
Current methods for RNA selection and enrichment, particularly for mRNA molecules, are inefficient, species-limited, and costly, often requiring multiple rounds of depletion and struggling with the removal of high-copy rRNA and tRNA, especially in non-model organisms.
Innovation Solution
The use of IFIT proteins from the Interferon-Induced Proteins with Tetratricopeptide Repeats family, which specifically bind to RNA molecules with specific 5' end modifications, allowing for the selective enrichment of mRNA molecules from diverse organisms without the need for species-specific probes or extensive depletion rounds.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If commercial rRNA removal kits with species-specific probes are used, then rRNA depletion efficiency is improved for model organisms, but the method becomes limited to specific species and requires multiple successive depletion rounds for non-model organisms
Solution Approach 1:
The patent employs poly(A) polymerase enzyme that universally catalyzes poly(A) tail addition to RNA molecules across all species, replacing species-specific probes with a universal enzymatic mechanism that functions across diverse organisms including bacteria, archaea, and eukaryotes
Solution Approach 2:
The invention changes the biochemical parameter from sequence-specific hybridization to enzymatic activity-based modification, where poly(A) polymerase recognizes and modifies RNA based on structural features rather than species-specific sequences, enabling broad applicability
2Manufacturing precision
If multiple successive depletion rounds are performed, then rRNA removal completeness is improved for non-model organisms, but the time required and cost increase significantly
Solution Approach 1:
The patent performs preliminary poly(A) tail addition to all RNA molecules before sequencing library preparation, creating a universal marker that enables single-step enrichment rather than requiring multiple sequential depletion rounds, thus saving time while achieving complete rRNA removal
Solution Approach 2:
The invention extracts and removes rRNA-containing fractions after poly(A) tail addition, separating target mRNA (which retains poly(A) tail) from rRNA (which loses poly(A) tail during the process), achieving complete removal in a single operation
3Manufacturing precision
If poly(A) tail-based selection is used, then mRNA enrichment is improved for eukaryotic organisms, but the method fails to work for prokaryotic organisms and certain eukaryotic mRNAs without poly(A) tails
Solution Approach 1:
The patent uses poly(A) polymerase to add poly(A) tails universally to all RNA molecules regardless of organism type or original poly(A) status, creating a universal enrichment strategy that works for both prokaryotes and eukaryotes, including mRNAs that naturally lack poly(A) tails
Solution Approach 2:
Instead of selecting for existing poly(A) tails (which excludes prokaryotes and some eukaryotic mRNAs), the invention inverts the approach by adding poly(A) tails to all RNA molecules and then removing those that lose the tail (rRNA), thereby enriching for mRNA across all organism types
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 method provides a universal, fast, and cost-effective means to enrich mRNA molecules, reducing the rRNA and tRNA contamination, and is applicable to a wide range of species, including bacteria and lower eukaryotes, with minimal equipment requirements and no introduction of RNA modifications, enhancing the sensitivity and selectivity of molecular analyses.
Implementation Method 1
The invention uses a poly(A) polymerase to add poly(A) tails to all RNA molecules in the sample, creating a universal marker that works across all species including bacteria, archaea, and eukaryotes
Implementation Method 2
rRNA-containing fractions are removed after poly(A) tail addition, separating target mRNA from rRNA based on their differential response to the enzymatic treatment
Data Source
AI summary
A method for RNA selection and/or enrichment, especially with mRNA molecules, from a pool of RNA molecules, the method comprising a step of incubating a sample containing a pool of RNA molecules with an RNA binding protein to form RNA-RNA binding protein complexes, wherein a protein of IFIT family of proteins or its functional variants, homologues or mutants are used as the RNA binding protein. The RNA molecule selected and/or enriched by the method and is used for detection in RNA pathogen-based diagnostic tests and for preparation of libraries for RNA sequencing.