Microbial Single-Cell RNA Sequencing via In-Cell Polyadenylation
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Solution Overview
Problem
Current methods for single-cell RNA sequencing (scRNA-seq) in bacteria face challenges due to low mRNA content, lack of polyadenylation, diverse cell walls and membranes, and small size, which hinder effective labeling and isolation of bacterial cells.
Innovation Solution
The development of microSPLiT (Microbial Split-Pool Ligation Transcriptomics) methods and kits for uniquely labeling nucleic acid molecules within bacterial cells, involving fixation, permeabilization, reverse transcription, and combinatorial labeling of cDNA, using enzymes like lysozyme and poly(A) polymerase to enrich mRNA and maintain cell integrity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If eukaryotic scRNA-seq methods are directly applied to bacteria, then comprehensive gene expression profiling can be achieved, but the low mRNA content and lack of polyadenylation in bacteria make mRNA separation and labeling ineffective
Solution Approach 1:
The patent modifies the biochemical parameters of bacterial mRNA by performing in-cell polyadenylation using poly(A) polymerase. This adds poly(A) tails to bacterial mRNA molecules, changing their physical-chemical properties to enable specific capture by oligo(dT) reverse transcription primers, thereby resolving the contradiction between low mRNA content and effective mRNA separation/labeling
Solution Approach 2:
The patent introduces poly(A) polymerase as an intermediary enzyme that acts on bacterial mRNA within intact cells. This mediator converts non-polyadenylated bacterial mRNA into polyadenylated mRNA, enabling subsequent specific binding to oligo(dT) primers and successful reverse transcription, thus bridging the gap between bacterial mRNA characteristics and eukaryotic scRNA-seq methodology
2Ease of operation
If bacterial cells are subjected to lysis or permeabilization for nucleic acid access, then RNA can be extracted, but diverse cell walls and membranes interfere with effective permeabilization
Solution Approach 1:
The patent performs reverse transcription and polyadenylation steps while bacterial cells remain intact, before any lysis or permeabilization. This preliminary action allows enzymatic reactions to occur inside intact cells, avoiding the need to overcome diverse cell wall and membrane barriers during critical biochemical steps
Solution Approach 2:
The patent utilizes the bacterial cell's own metabolic machinery by performing reverse transcription and polyadenylation in situ within intact cells. The cells themselves provide the necessary environment and cofactors for these reactions, eliminating the need for external intervention that would require breaking the cell wall or membrane
3Measurement precision
If small bacterial cells are isolated using microfluidic methods, then single-cell analysis can be performed, but the small size hinders effective isolation and sorting
Solution Approach 1:
The patent combines multiple bacterial cells into aggregates that maintain single-cell transcriptional identity. By performing reverse transcription and polyadenylation on intact cells (whether individual or in aggregates) and using combinatorial indexing, the method achieves single-cell resolution without requiring physical isolation of individual small bacterial cells through microfluidics
Solution Approach 2:
The patent applies combinatorial indexing where each cell in an aggregate is assigned a unique molecular barcode through sequential rounds of labeling. This segmentation of the identification process allows tracking of individual cell transcripts even when cells are physically aggregated, effectively decoupling isolation requirements from single-cell analysis capability
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 comprehensive gene expression profiling of bacterial cells by effectively dissociating aggregates, enriching mRNA, and uniquely labeling cDNA, allowing for the identification of rare cell states and transcriptional pathways in bacterial populations.
Implementation Method 1
cell wall-degradation enzyme
Implementation Method 2
poly(A) polymerase to enrich mRNA
Implementation Method 3
reverse transcribing mRNA within the plurality of microbial cells to provide cDNA
Data Source
AI summary
Methods and kits for uniquely labeling nucleic acid molecules within a plurality of microbial cells are described. In an embodiment, the method comprises fixing and permeabilizing the plurality of microbial cells; dissociating microbial cell aggregates within a suspension comprising the plurality of microbial cells; reverse transcribing mRNA within the plurality of microbial cells to provide cDNA; and combinatorially labelling the cDNA to provide labelled cDNA.