Antibody-Targeted Tagmentation for In Situ Chromatin RNA Profiling

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Solution Overview

Problem

Existing immunoprecipitation-based methods for mapping cellular RNAs require large sample inputs and optimized cross-linking conditions, necessitating the development of sensitive in situ technologies that do not rely on cross-linking or immunoprecipitation.

Innovation Solution

An in-situ method using a tethered enzyme complex for mapping cellular RNA, involving binding a first recognition agent to RNA or its associated proteins, tethering a transposase fused to protein A (pA-transposase) for reverse transcription and tagmentation, and preparing sequencing libraries of the RNA/DNA hybrid.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Measurement precision

If immunoprecipitation-based methods are used to map cellular RNAs, then RNA-protein and RNA-chromatin interactions can be detected, but large sample inputs and optimized cross-linking conditions are required

Engineering Contradiction:

Improvedetection sensitivity of RNA interactionsVSAvoidsample input requirement

Core Design Contradiction:

Measurement precisionVSQuantity of substance

Solution Approach 1:

The patent replaces the mechanical immunoprecipitation system with an enzymatic tagmentation system. Instead of using antibodies to pull down RNA-protein complexes, the method uses a transposase enzyme that simultaneously fragments DNA and tags it with sequencing adapters. This enzymatic approach eliminates the need for cross-linking and immunoprecipitation, enabling sensitive detection of chromatin-associated RNAs with reduced sample input requirements

Inventive Principle:

Principle #28Mechanics substitution (Replace mechanical system)

Solution Approach 2:

The patent changes the fundamental parameters of the methodology by shifting from cross-linking-based immunoprecipitation to enzyme-based tagmentation. This parameter change allows the system to operate without cross-linking conditions, reducing the sample input requirement while maintaining detection sensitivity for RNA-chromatin interactions

Inventive Principle:

Principle #35Parameter changes

2Measurement precision

If immunoprecipitation-based methods are used to map cellular RNAs, then RNA interactions can be profiled, but the process is time-consuming and requires complex optimization

Engineering Contradiction:

Improveprofiling accuracy of RNA interactionsVSAvoidprotocol time and optimization time

Core Design Contradiction:

Measurement precisionVSLoss of time

Solution Approach 1:

The patent replaces the multi-step immunoprecipitation process with a single enzymatic tagmentation reaction. The transposase enzyme simultaneously performs DNA fragmentation and adapter tagging in one step, eliminating the need for separate cross-linking, immunoprecipitation, and purification steps. This substitution dramatically reduces protocol time while maintaining profiling accuracy

Inventive Principle:

Principle #28Mechanics substitution (Replace mechanical system)

Solution Approach 2:

The patent performs the tagmentation reaction in situ within intact nuclei, eliminating the need for subsequent purification and resuspension steps. By preparing the sequencing libraries directly in the nuclear environment, the method avoids time-consuming intermediate steps and reduces overall protocol time

Inventive Principle:

Principle #10Preliminary action

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

Enables efficient and sensitive mapping of chromatin-associated RNAs with reduced sample requirements, suitable for rapid low-cost profiling of RNA-protein interactions and RNA-chromatin interactions.

Implementation Method 1

the second recognition agent is conjugated to a biotin-binding moiety; tethering at least one molecule required for reverse transcription comprising a biotinylated oligonucleotide for cDNA synthesis priming and for PCR amplification to the second recognition agent

Methodology Applied

Scientific EffectBiotin-streptavidin interaction:

Implementation Method 2

allowing the at least one molecule required for reverse transcription to convert a mature transcript near the binding site of the first recognition agent to an RNA/DNA hybrid comprising a first sequencing adapter sequence and a priming sequence

Methodology Applied

Scientific EffectReverse transcription:

Implementation Method 3

allowing the pA-transposase to tagment the RNA/DNA hybrid; and preparing sequencing libraries of the RNA/DNA hybrid

Methodology Applied

Scientific EffectTagmentation:

Implementation Method 4

tethering a transposase fused to protein A (pA-transposase) comprising a first sequencing adapter sequence to the first recognition agent and the second recognition agent

Methodology Applied

Scientific EffectProtein A binding:

Data Source

PatentUS20250263782A1Profiling RNA at chromatin targets in situ by antibody-targeted tagmentation

Publication Date: 2025.08.21 FRED HUTCHINSON CANCER CENT

AI summary

Embodiments of the present disclosure provide methods and kits for mapping a cellular RNA using a tethered enzyme complex. Embodiments of the method comprise binding a first recognition agent to an epitope of RNA or its associated proteins; binding a second recognition agent that specifically binds to the first recognition agent; and tethering an enzyme complex to the first recognition agent and/or the second recognition agent, wherein the enzyme complex enables reverse transcription to convert a mature transcript near the binding site of the first recognition agent to an RNA/DNA hybrid and tagmentation of the RNA/DNA hybrid for use in preparing sequencing libraries of the RNA/DNA hybrid.