Spatial Aptamer Co-Assays for mRNA-Protein Signal Co-Localization
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Solution Overview
Problem
Existing spatial assays struggle to co-localize mRNA and protein signals effectively, as commercially available methods are limited in detecting proteins and face challenges such as protein binding interference, aptamer removal difficulty, and protein abundance dynamic range issues.
Innovation Solution
Aptamers are used to prepare spatial proteome sequencing libraries by associating with proteins, removing unbound aptamers, and releasing target nucleotide sequences for capture, with methods involving ultraviolet radiation, enzymes, or chemical cleavage to address these challenges.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If a long sequence is added to the aptamer to enable capture on the barcoded surface, then capture capability is improved, but protein binding by the aptamer is impacted
Solution Approach 1:
The aptamer is divided into two functional segments: a capture sequence (5'-terminal) that binds to the barcoded surface and a protein-binding sequence (3'-terminal) that interacts with the target protein. This segmentation allows each part to perform its specific function independently, resolving the conflict between capture capability and protein binding affinity.
Solution Approach 2:
The aptamer acts as an intermediary molecule between the barcoded surface and the target protein. It simultaneously interacts with both the surface (via capture sequence) and the protein (via protein-binding sequence), enabling indirect capture without requiring the capture sequence to directly bind the protein.
2Difficulty of detecting and measuring
If aptamers are used to detect proteins in situ, then detection capability is improved, but aptamers are difficult to remove from their target proteins
Solution Approach 1:
A cleavable linkage is pre-installed between the aptamer and the barcoded surface. After the aptamer binds to the protein, a cleavage agent is applied to break the linkage, releasing the aptamer-protein complex from the surface. This preliminary preparation of the cleavable linkage enables controlled release without requiring forceful removal methods.
Solution Approach 2:
The cleavable linkage is designed to be stable under normal assay conditions but can be broken by specific cleavage agents (chemical, enzymatic, or photolytic). By changing the chemical or physical parameters of the environment, the linkage can be selectively broken to release the aptamer from the surface.
3Productivity
If free aptamers are not prevented from binding to the barcoded surface, then capture efficiency is improved, but non-specific binding increases
Solution Approach 1:
Unbound aptamers are removed from the solution through washing steps before the capture process. This extraction of free aptamers prevents them from non-specifically binding to the barcoded surface, ensuring that only aptamers that have bound to their target proteins are captured.
Solution Approach 2:
The aptamer sequence is copied and amplified after capture to generate sufficient signal for detection. This copying process allows the system to work with low-abundance target proteins while maintaining specificity, as the amplification occurs after specific binding has already occurred.
4Measurement precision
If proteins are detected directly, then detection sensitivity is improved, but the large dynamic range of protein abundance makes quantification difficult
Solution Approach 1:
The aptamer sequence is copied and amplified after capture to generate sufficient signal for detection. This copying process allows the system to work with low-abundance target proteins while maintaining specificity, as the amplification occurs after specific binding has already occurred.
Solution Approach 2:
The aptamer acts as an intermediary that bridges the gap between the target protein and the detection system. It binds to the protein with high affinity and then provides a detectable signal through its sequence, enabling sensitive detection across a wide dynamic range of protein abundances.
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
The method enables efficient co-localization of mRNA and protein signals, overcoming protein binding interference and dynamic range issues, facilitating better understanding of gene expression and disease mechanisms.
Implementation Method 1
contacting a plurality of aptamers to the biological sample on the surface, the contacting resulting in association of individual aptamers in the plurality of aptamers with individual proteins in the biological sample
Implementation Method 2
the plurality of aptamers is cleaved via ultraviolet radiation, an enzyme, or chemical cleavage
Implementation Method 3
the plurality of aptamers is cleaved via ultraviolet radiation, an enzyme, or chemical cleavage
Implementation Method 4
the plurality of aptamers is cleaved via ultraviolet radiation, an enzyme, or chemical cleavage
Implementation Method 5
each capture oligonucleotide in the plurality of capture oligonucleotides comprises (i) a capture nucleotide sequence at the 3′ end that is configured to bind to a target nucleotide sequence
Data Source
AI summary
The present disclosure relates, in general, to methods of preparing a spatial proteome and/or transcriptome sequencing library. The spatial proteome and/or transcriptome sequencing library from a biological sample is useful, in some aspects, to determine a genetic profile and help diagnose a subject who has or is at risk of having a disorder, and improve treatment of the subject.