PLUMAGE Assay for Full-Length 5' UTR Mutation Analysis
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current methods for studying the functional consequences of 5' untranslated region (UTR) mutations in cancer are limited by their inability to analyze full-length 5' UTRs and simultaneously assess mRNA transcript levels and translation efficiency.
Innovation Solution
The development of the PLUMAGE (Pooled full-length UTR Multiplex Assay on Gene Expression) method, which combines long-read and short-read sequencing technologies to analyze target nucleic acid sequences, allowing for the precise quantification of effects of patient-based somatic mutations on mRNA transcript levels and translation efficiency within full-length 5' UTRs.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If traditional MPRAs are used to study UTR elements, then high-throughput characterization of regulatory elements is achieved, but analysis is limited to short genomic regions less than 200 bases
Solution Approach 1:
The patent segments the analysis into two distinct sequencing approaches: long-read sequencing (PacBio) to capture full-length 5' UTR sequences and associate them with barcodes, and short-read sequencing (Illumina) to precisely quantify transcript and translation levels. This segmentation allows each sequencing technology to operate in its optimal range while collectively achieving comprehensive full-length UTR analysis that neither technology could accomplish alone.
2Measurement precision
If current methods are used to study 5' UTR mutations, then some functional consequences are identified, but simultaneous assessment of mRNA transcript levels and translation efficiency is not achieved
Solution Approach 1:
The patent creates a universal assay platform (PLUMAGE) that simultaneously performs multiple functions: it characterizes full-length 5' UTR sequences, quantifies mRNA transcript levels, and measures translation efficiency in a single integrated experiment. The barcoded reporter constructs enable all three measurements to be performed concurrently across hundreds of 5' UTR variants, providing comprehensive multi-layer gene expression assessment that reveals how mutations affect both transcription and translation.
3Length of stationary object
If full-length 5' UTRs are analyzed, then comprehensive functional interrogation is achieved, but traditional sequencing technologies cannot capture the full length due to read length limitations
Solution Approach 1:
The patent introduces barcoded reporter constructs as intermediaries that bridge long-read and short-read sequencing technologies. The barcodes serve as unique identifiers that link full-length 5' UTR sequences (captured by long-read sequencing) with their corresponding transcript and translation measurements (quantified by short-read sequencing). This intermediary approach enables the integration of both sequencing modalities to achieve comprehensive full-length UTR analysis.
Data Source
AI summary
A method for analyzing an ability of target nucleic acid sequences to impact gene expression is described. In an embodiment, the method includes cloning the target nucleic acid sequences and associated barcode nucleic acid sequences into a plurality of plasmids, sequencing the plasmids to provide long-read sequencing information based on a target nucleic acid sequence of the target nucleic acid sequences and an associated barcode nucleic acid sequence, associating the target nucleic acid sequence with the associated barcode nucleic acid sequence based on the long-read sequencing information, transducing the plurality of plasmids into a plurality of cells, extracting DNA, total mRNA, and polysome-bound mRNA from the cells, sequencing the barcode nucleic acid sequences in the extracted DNA, total mRNA, and polysome-bound mRNA to provide short-read sequencing information, and analyzing the target nucleic acid sequences by comparing the long-read sequencing information and the short-read sequencing information.