DNA Probes for Depleting Abundant Small Noncoding RNA
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Solution Overview
Problem
Current methods for depleting off-target RNA from nucleic acid samples are inefficient, requiring high sample input, technician time, and cost, and often fail to effectively remove abundant small noncoding RNA species, which interferes with the analysis of target RNA in samples like FFPE tissues, leading to wasted sequencing reads and duplicative results.
Innovation Solution
A method using a probe set comprising DNA probes complementary to discontiguous sequences of off-target RNA molecules, specifically targeting RN7SK, RN7SL1, RN7SL2, RN7SL5P, RPPH1, and SNORD3A, which hybridize with off-target RNA and are then degraded by a ribonuclease, allowing for the depletion of these molecules from the sample.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If current commercially available methods (RiboZero, NEBNext) are used to deplete off-target RNA, then some RNA depletion is achieved, but the methods require high sample input, extensive technician hands-on time, and high cost while failing to effectively deplete abundant small noncoding RNA
Solution Approach 1:
The patent introduces an automated liquid handling system as an intermediary between the researcher and the RNA depletion process. The system uses robotic liquid handling to automatically perform probe hybridization and RNase treatment, eliminating the need for manual technician operations while achieving superior depletion of small noncoding RNA compared to commercial methods
Solution Approach 2:
The patent changes the operational parameters by using automated liquid handling with precise control over reaction conditions, incubation times, and reagent addition. This automation enables the use of specific probe designs and RNase treatment protocols that are not practical with manual methods, achieving more effective depletion of abundant small noncoding RNA
2Reliability
If current depletion methods are used, then some off-target RNA is removed, but abundant small noncoding RNA remains which causes wasted sequencing reads and duplicative results
Solution Approach 1:
The patent segments the depletion approach by using multiple distinct probes targeting different small noncoding RNA species (RN7SK, RN7SL1, RN7SL2, RN7SL5P, RPPH1, SNORD3A) rather than relying on a single commercial kit. The automated system ensures each probe is properly delivered and hybridized, enabling comprehensive depletion across multiple RNA types that manual methods cannot effectively address
Solution Approach 2:
The automated liquid handling system performs the depletion process autonomously without requiring technician intervention. The system automatically adds probes, controls hybridization conditions, and applies RNase treatment, enabling a more thorough and consistent depletion of small noncoding RNA that would be difficult to achieve manually while reducing human error and variability
3Ease of manufacture
If manual depletion methods are used, then the process can be performed with standard equipment, but it requires high sample input and extensive technician hands-on time
Solution Approach 1:
The patent introduces an automated liquid handling system as an intermediary that bridges standard laboratory equipment with high-throughput automation. The system uses robotic liquid handling to perform all depletion operations, maintaining compatibility with standard lab equipment while dramatically increasing productivity by eliminating manual operations and enabling parallel processing of multiple samples
Solution Approach 2:
The automated system performs preliminary actions by pre-programming the depletion protocol with optimized probe concentrations, hybridization times, and RNase treatment conditions. This pre-programming enables the system to execute the depletion process efficiently without requiring real-time technician decisions, thereby increasing productivity while maintaining compatibility with standard laboratory equipment
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 significantly reduces the presence of off-target RNA, increasing the availability of target RNA for analysis, thereby enhancing the detection of medically actionable information and reducing sequencing costs and hands-on time.
Implementation Method 1
contacting a nucleic acid sample comprising at least one target RNA or DNA sequence and at least one off-target RNA molecule with the probe set, thereby hybridizing the DNA probes to the at least one off-target RNA molecule to form DNA:RNA hybrids
Implementation Method 2
contacting the DNA:RNA hybrids with a ribonuclease that degrades the RNA from the DNA:RNA hybrids, thereby degrading the off-target RNA molecules in the nucleic acid sample to form a degraded mixture
Data Source
AI summary
Described herein are methods for depleting library fragments prepared from off-target RNA sequences. Libraries enriched or depleted with the present methods may be used for sequencing. Also described are probes and methods for depletion or supplementing depletion of off-target RNA from human and non-human samples.