Deterministic Barcoding of Fragmented RNA for FFPE Spatial Profiling
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Solution Overview
Problem
Existing technologies face challenges in analyzing genomic information from clinically archived formalin-fixed paraffin-embedded (FFPE) tissues due to RNA fragmentation and degradation, limiting the ability to conduct comprehensive spatial transcriptomic and proteomic analyses.
Innovation Solution
The Patho-DBiT method employs in situ polyadenylation and deterministic barcoding using microfluidic devices to add poly(A) tails to fragmented and naturally RNA molecules, enabling the production of spatially barcoded cDNAs for mapping and sequencing.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Duration of action of stationary object
If RNA molecules are stored in FFPE tissues for long-term archiving, then tissue preservation and availability are improved, but RNA fragmentation and degradation occur, resulting in loss of analyzable genomic data
Solution Approach 1:
The patent applies preliminary action by performing polyadenylation of fragmented RNA molecules before conventional RNA analysis. The polyadenylate polymerase adds poly(A) tails to the 3' ends of fragmented RNA molecules in situ within the FFPE tissue sections, converting them into analyzable forms that can be subsequently processed through reverse transcription and spatial barcoding. This preliminary modification restores the analytical utility of degraded RNA without requiring tissue resection or alternative storage approaches.
2Ease of manufacture
If conventional RNA analysis methods are used on FFPE tissues, then analysis simplicity is maintained, but the ability to capture fragmented and degraded RNA is limited
Solution Approach 1:
The patent employs an intermediary approach by introducing polyadenylate polymerase as a mediating enzyme that modifies fragmented RNA molecules in situ. This intermediary enzyme adds poly(A) tails to the 3' ends of fragmented RNA, making them suitable for conventional reverse transcription and sequencing workflows. The polyadenylation step acts as a bridge between the degraded RNA state and the requirements of standard analysis methods, enabling broad RNA species coverage while maintaining workflow simplicity.
Solution Approach 2:
The patent applies parameter changes by modifying the chemical state of RNA molecules through polyadenylation. The polyadenylate polymerase changes the 3' end parameters of fragmented RNA molecules by adding poly(A) tails, thereby altering their structural and functional properties to make them compatible with conventional analysis methods. This chemical modification enables the capture of fragmented and degraded RNA species that would otherwise be inaccessible to standard workflows.
3Measurement precision
If polyadenylation is performed in situ within tissue sections, then spatial information is preserved, but additional reagents and steps are required
Solution Approach 1:
The patent applies segmentation by dividing the tissue section into multiple regions served by separate microfluidic devices. The first microfluidic device delivers polyadenylation reagents to specific regions, while the second microfluidic device delivers reverse transcription and barcoding reagents to the same or different regions. This segmentation allows independent optimization of each step and enables parallel processing of multiple tissue sections or regions, managing complexity through modular design.
Solution Approach 2:
The patent employs universality by designing a multi-functional microfluidic platform that can perform polyadenylation, reverse transcription, and spatial barcoding in a single integrated system. The device incorporates multiple reagent delivery channels and reaction chambers that can accommodate different enzymatic reactions sequentially or in parallel, eliminating the need for separate processing steps and reducing overall experimental complexity while maintaining spatial resolution.
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 approach allows for high-sensitivity spatial transcriptomics, capturing a broad spectrum of RNA species, including fragmented mRNAs, non-coding RNAs, and splicing isoforms, thereby overcoming the limitations of FFPE tissues and providing valuable insights into RNA biology and disease development.
Implementation Method 1
first polyadenylating RNA molecules, such as fragmented RNA molecules, which lack a poly(A) tail
Implementation Method 2
delivering reverse transcription reagents to the tissue section
Implementation Method 3
delivering to the tissue section a first set of barcoded polynucleotides, a second set of barcoded polynucleotides, and ligation reagents to produce spatially barcoded cDNAs
Data Source
AI summary
The disclosure relates to compositions and methods for spatial whole transcriptome sequencing in processed tissues.