SPIDR Antibody-Bead Conjugate Pool for Multiplexed RBP-RNA Mapping
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Solution Overview
Problem
Current methods for mapping RNA binding proteins (RBPs) are limited to studying a single RBP at a time, requiring significant resources and are not applicable to diverse cell types or rare cell populations, leading to incomplete and cell-type-specific RBP binding maps.
Innovation Solution
The Split and Pool Identification of RBP targets (SPIDR) method uses antibody-bead conjugate pools and split-and-pool barcoding to simultaneously map dozens to hundreds of RBPs across various RNA types in a single experiment, enabling high-resolution, transcriptome-wide RBP-RNA interaction maps.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If CLIP-seq method is used to map RBP-RNA interactions, then measurement precision is improved, but productivity deteriorates due to single-RBP limitation
Solution Approach 1:
The method segments the RBP mapping problem by assigning unique molecular barcodes to each RBP of interest. These barcodes are incorporated into antibody-bead conjugates, allowing simultaneous immunoprecipitation and identification of multiple RBPs in a single experiment while maintaining the precision of individual RBP mapping through barcode-specific sequencing analysis
Solution Approach 2:
The protocol creates a universal platform that can map any number of RBPs simultaneously by using a common set of reagents and procedures. The antibody-bead conjugate pool with unique barcodes serves multiple RBPs at once, and the single CLIP-seq experiment generates data for all targeted RBPs, making the method universally applicable to diverse RBP studies
2Measurement precision
If multiple individual CLIP-seq experiments are performed to map hundreds of RBPs, then measurement precision is maintained, but loss of time increases significantly
Solution Approach 1:
The method merges multiple individual RBP mapping experiments into a single multiplexed experiment. By combining antibody-bead conjugates with unique barcodes for different RBPs into a single pool and performing one CLIP-seq experiment, the protocol simultaneously captures RBP-RNA interactions for all targeted RBPs, reducing time loss while preserving mapping accuracy through barcode-specific data analysis
3Measurement precision
If large number of cells are used for individual RBP mapping, then measurement precision is improved, but productivity deteriorates due to resource requirements
Solution Approach 1:
The method segments the cell resource requirement by using unique molecular barcodes to tag each RBP's immunoprecipitated RNA. This allows the signal from a limited number of cells to be partitioned and attributed to specific RBPs through barcode identification in sequencing data, maintaining detection accuracy while reducing the total cell input needed compared to performing separate experiments for each RBP
4Measurement precision
If cell-type-specific RBP maps are generated from individual experiments, then measurement precision for that cell type is improved, but adaptability deteriorates across different cell types
Solution Approach 1:
The protocol establishes a universal methodology that can be applied to any cell type by simply changing the antibody targets. The same barcode-based immunoprecipitation and sequencing workflow maintains measurement precision for cell-type-specific binding while enabling adaptation to diverse cell types, including primary cells and disease models, through the flexibility of selecting different RBP-specific antibodies
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
SPIDR dramatically reduces the number of cells needed for RBP mapping, provides comprehensive RBP binding information across cell types, and generates accurate single nucleotide contact maps, facilitating the exploration of RBP dynamics and their roles in diverse biological processes.
Implementation Method 1
each antibody-bead conjugate in an antibody-bead conjugate population comprises an antibody specific for a single RNA binding protein
Implementation Method 2
CLIP works by utilizing UV light to covalently crosslink RNA and directly interacting proteins
Data Source
AI summary
Aspects of the present disclosure generally relate to methods and compositions for detecting an association between a RNA binding protein and a RNA. Some aspects of the present disclosure relate to methods of generating an antibody-bead conjugate pool. Some aspects of the present disclosure relate to kits and compositions for performing the methods disclosed herein.