Chimeric RNA Editing Proteins for Translation Rate Sequencing
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Solution Overview
Problem
Current techniques for measuring translation efficiency, such as ribosome profiling and polysome profiling, require the removal of ribosomal RNA due to unwanted contamination, which complicates the process.
Innovation Solution
The use of chimeric proteins comprising an RNA binding protein and an RNA editing protein to introduce nucleotide substitutions into target mRNA, allowing for sequencing-based detection of ribosome initiation, scanning, and release, thereby determining relative translation rates and binding sites.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If ribosome profiling or polysome profiling is used to measure translation efficiency, then translation rate information can be obtained, but ribosomal RNA must be removed due to contamination which complicates the process
Solution Approach 1:
The patent introduces an intermediary system consisting of chimeric proteins that fuse RNA binding domains (specifically ribosomal protein domains) with RNA editing domains (adenosine deaminase). This intermediary system allows indirect measurement of translation efficiency by using the RNA editing activity as a proxy for ribosome occupancy, thereby avoiding the need for direct ribosomal RNA isolation and the complex associated procedures
Solution Approach 2:
The patent replaces the mechanical/biochemical separation process (ribosomal RNA removal through centrifugation and purification steps) with a molecular biochemical assay (RNA editing followed by sequencing). Instead of physically separating ribosomal components, the method uses enzymatic modification of RNA that is protected by ribosomes, substituting a complex mechanical separation system with a simpler enzymatic labeling system
2Reliability
If ribosomal RNA removal is performed to eliminate contamination, then measurement accuracy improves, but the process becomes more complex and time-consuming
Solution Approach 1:
The patent performs preliminary action by introducing the chimeric protein system into cells before the actual measurement is needed. The chimeric proteins continuously edit RNA in a translation-dependent manner, so when RNA is extracted and sequenced, the editing patterns are already established and directly reflect ribosome occupancy without requiring any preliminary ribosomal RNA removal steps
3Measurement precision
If traditional ribosome profiling is used, then translation efficiency can be measured, but ribosomal RNA contamination requires additional purification steps
Solution Approach 1:
The chimeric protein acts as an intermediary that translates ribosome occupancy information into a detectable RNA modification signal. The RNA binding domain of the chimeric protein binds to RNA in a translation-dependent manner, and the RNA editing domain modifies the bound RNA, creating an indirect but easily detectable signal that avoids the need for direct ribosomal RNA handling and purification
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
Enables accurate and efficient determination of translation rates and RNA binding protein binding sites without the need for ribosomal RNA removal, providing a reliable surrogate measure for ribosome activity.
Implementation Method 1
an RNA editing protein; determining a plurality of nucleotide substitutions introduced into the target mRNA by the RNA editing protein
Data Source
AI summary
Provided are methods for using chimeric proteins to produce RNA modifications that can be detected by sequencing methods, including methods detecting relative translation rates of various mRNAs. Also provided herein are compositions comprising chimeric proteins, wherein the chimeric proteins comprise a RNA editing protein and a ribosomal protein.