Spatial Transcriptomics Library Prep for Fragmented Tissue RNA
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Solution Overview
Problem
Current spatial transcriptomics workflows face challenges in capturing and converting mRNA from frozen or FFPE tissue samples due to fragmentation, degradation, and crosslinking, resulting in low quality and quantity of RNA and DNA, leading to poor mRNA transcript mapping rates.
Innovation Solution
A method involving in situ polyadenylation of RNA using polynucleotide kinase and polyadenylate polymerase to modify 3' phosphate groups, followed by capture on oligo-dT substrates, and subsequent cDNA synthesis with high processivity reverse transcriptases to improve mRNA capture and library preparation.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Stability of the object's composition
If tissue samples are frozen or fixed with formalin for preservation, then sample stability and preservation are improved, but RNA fragmentation, degradation and crosslinking occur resulting in low quality and quantity of RNA
Solution Approach 1:
The patent applies preliminary action by performing end repair of RNA fragments before polyadenylation. The 3' phosphate groups on fragmented RNA are converted to 3' hydroxyl groups using polynucleotide kinase, creating compatible ends for subsequent polyadenylation. This preliminary repair step enables efficient polyA tail addition to otherwise unusable fragmented RNA from FFPE samples.
Solution Approach 2:
The patent changes the chemical parameter of the RNA 3' end by converting phosphate groups to hydroxyl groups through enzymatic treatment with polynucleotide kinase. This parameter change makes the RNA ends chemically compatible for polyadenylation, transforming degraded RNA fragments into molecules that can be efficiently captured and converted to cDNA.
2Productivity
If polyadenylation is performed on fragmented RNA, then capture efficiency on oligo-dT surface is improved, but standard workflows fail to efficiently capture FFPE RNA due to 3' phosphate groups
Solution Approach 1:
The patent performs preliminary enzymatic treatment with polynucleotide kinase to convert 3' phosphate groups to 3' hydroxyl groups before polyadenylation. This preliminary action removes the barrier that prevents efficient oligo-dT capture, enabling reliable mRNA enrichment from FFPE samples that would otherwise fail with standard workflows.
Solution Approach 2:
The patent introduces an intermediary enzymatic step using polynucleotide kinase as a mediator between the fragmented RNA and the polyadenylation reaction. This intermediary treatment modifies the RNA ends to be compatible with polyA tail addition, bridging the gap between degraded RNA and efficient oligo-dT capture.
3Manufacturing precision
If standard cDNA synthesis methods are used, then library preparation can proceed, but cDNA fragment length is short resulting in poor alignment to exonic regions
Solution Approach 1:
The patent performs preliminary polyadenylation of repaired RNA fragments before cDNA synthesis. By adding polyA tails to the 3' ends of fragmented RNA, the patent creates uniform, extended templates that enable high processivity reverse transcriptases to synthesize longer cDNA fragments with improved alignment quality to exonic regions.
Solution Approach 2:
The patent applies partial action by performing polyadenylation only on the 3' ends of RNA fragments rather than attempting to repair entire fragments. This targeted modification at the 3' end is sufficient to enable extended cDNA synthesis and improve alignment quality without requiring complete RNA reconstruction.
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
Enhances mRNA capture and library conversion efficiency, resulting in longer cDNA fragments and improved alignment to exonic mRNA transcript regions, increasing the mapping rate in RNA-seq alignment.
Implementation Method 1
contacting total RNA isolated from the sample with polynucleotide kinase (PNK) to modify 3' phosphate to a hydroxyl group
Implementation Method 2
contacting the end repaired total RNA with polyadenylate polymerase (PAP) and adenosine nucleotides to generate polyadenylated total RNA
Implementation Method 3
capturing the polyadenylated total RNA on a substrate comprising one or more oligonucleotides comprising poly T sequences
Data Source
AI summary
The present disclosure relates, in general, to materials and methods for improving RNA capture in situ from tissue samples and improved methods for synthesizing cDNA from the captured RNA.