MICTAG Probe Spatial Sequencing mRNA Resolution
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Solution Overview
Problem
Existing in situ sequencing methods face limitations in the number of measurable mRNA sequences and expression dynamics due to signal amplification issues and poor RNA capture efficiency, leading to impaired signal discrimination and poor spatial resolution, especially in single-cell analysis.
Innovation Solution
The method employs optical coding using MICTAG probes that bind to mRNA, allowing reverse transcription and rolling circle amplification, enabling high-resolution single-cell mRNA profiling and sequencing by incorporating a mismatch code that can be read to determine the spatial location and sequence information of mRNA molecules.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If rolling circle amplification is used to amplify mRNA signals in situ, then signal detection sensitivity is improved, but the number of measurable mRNA sequences is limited due to cell size constraints and signal discrimination is impaired
Solution Approach 1:
The probe is divided into multiple functional segments: a binding region that hybridizes to target mRNA, a gap region that is filled by reverse transcription, and a padlock region that enables circularization. This segmentation allows the probe to perform multiple functions within a compact structure, enabling signal amplification while maintaining the capacity to measure multiple mRNA sequences simultaneously
Solution Approach 2:
The invention transitions from measuring only spatial information to simultaneously measuring both spatial information and sequence information by adding a new dimension of measurement. The padlock probe structure enables this dual measurement capability by incorporating both location-coding elements and sequence-coding elements in its design
2Measurement precision
If in situ signal amplification is performed, then mRNA detection sensitivity is improved, but spatial resolution deteriorates due to the large size of amplification products
Solution Approach 1:
The padlock probe adopts a flexible, compact molecular structure that forms a thin film-like configuration when bound to mRNA. This compact structure maintains small physical dimensions compared to traditional amplification products, preserving spatial resolution while still enabling signal amplification through the padlock mechanism and rolling circle amplification
Solution Approach 2:
The probe design embeds multiple functional elements within a compact nested structure: the binding region nests the gap region, which in turn nests the padlock region. This nested arrangement maximizes functional capacity while minimizing the overall physical footprint, maintaining spatial resolution during amplification
3Productivity
If barcoded capturing spots are used for mRNA capture, then sequencing throughput is improved, but capture efficiency is restricted and spatial resolution is poor
Solution Approach 1:
The invention extracts the mRNA capture process from the tissue context and performs it in solution using freely diffusing padlock probes. This extraction eliminates the spatial constraints and efficiency limitations of surface-based capturing spots while maintaining the ability to perform high-throughput sequencing of recovered mRNAs
Solution Approach 2:
The padlock probe serves as an intermediary molecule that bridges mRNA and the sequencing process. It first captures mRNA through hybridization in solution, then undergoes reverse transcription and circularization to create a stable complex that can be recovered and sequenced, thereby enabling efficient capture and high-throughput sequencing
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 overcomes the limitations of existing methods by providing high-resolution, single-cell mRNA profiling and sequencing, allowing for precise determination of mRNA distribution and sequence information, including single nucleotide polymorphisms, with improved signal discrimination and increased throughput.
Implementation Method 1
hybridizing a first padlock probe to a complementary sequence of a mRNA molecule
Implementation Method 2
reverse transcriptase-mediated gap-filling (copying of mRNA sequence into padlock probe)
Implementation Method 3
circularization of the padlock probe by a DNA ligase
Implementation Method 4
allowing rolling circle amplification of the padlock probe
Data Source
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AI summary
The invention is directed to a method to obtain the spatial location and sequence information of at least a part of a RNA or cDNA strand (006) in a sample comprising the steps a. hybridizing a first detection probe oligonucleotide (204) comprising 50 - 1000 nucleotides with its 3' and/or 5' end to the complementary part of the at least one RNA or cDNA strand, wherein the detection probe oligonucleotide is partially hybridized to a bridge oligonucleotide (205) comprising 5 - 100 nucleotides wherein a gap region (206) capable of binding oligonucleotides is created b. filling the gap region (206) in part with 1 to 16 barcode oligonucleotides comprising 4 - 20 nucleotides, wherein the barcode oligonucleotides determine the spatial information of the RNA or cDNA strand in the sample c. partially hybridizing a second detection probe oligonucleotide (204') comprising 50 - 1000 nucleotides with its 3' and/or 5' end to the complementary part of the same or cDNA strand and with the respective other end to the bridge oligonucleotide (205) to create a circular template d. multiplying the circular template by a polymerase capable of rolling circle amplification into rolonies comprising a plurality of concatemers e. determining the sequence of nucleotides of the rolonies