RNA Depletion Probe Layout for Uniform Binding and Low Off-Targeting
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Existing RNA depletion probe sets suffer from limitations such as uniform probe spacing and melting temperatures, leading to poor binding and off-target hybridization, especially in highly degraded samples, which complicates the detection of rare mRNA transcripts.
Innovation Solution
Designing RNA depletion probes with non-uniform, apparently random spacing and melting temperatures within a predetermined range, using biophysical properties and reference RNA sequence data to minimize off-target binding, allowing for efficient hybridization and digestion by RNase H in a single step.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If probe sets are designed with uniform spacing along the target RNA, then complete deletion coverage is achieved, but probe binding performance deteriorates due to GC content variation, secondary structure, and length constraints
Solution Approach 1:
The patent applies local quality by allowing each probe to have optimized local characteristics (GC content, length, spacing) rather than enforcing uniform global properties. Probes are designed with variable spacing and individual optimization to account for local variations in target RNA sequence composition and secondary structure, thereby maintaining both coverage and binding reliability.
Solution Approach 2:
The patent changes multiple probe parameters simultaneously including spacing distance, GC content, and length to optimize performance. Rather than maintaining constant spacing, the design allows spacing to vary based on local RNA properties, and adjusts probe GC content and length to achieve target melting temperatures while avoiding regions with problematic secondary structures.
2Stability of the object's composition
If probe sets use fixed melting temperature ranges, then uniform binding behavior is achieved, but design flexibility is reduced leading to poor performance in regions with extreme GC content or secondary structure
Solution Approach 1:
The patent introduces dynamics by making probe design parameters flexible rather than fixed. The spacing between probes, their GC content, and lengths are dynamically adjusted based on local target RNA properties such as sequence composition and predicted secondary structure, while still maintaining overall binding uniformity through controlled melting temperature ranges.
Solution Approach 2:
The patent changes probe parameters (spacing, GC content, length) to adapt to different regions of the target RNA. This allows the probe set to maintain binding uniformity through controlled Tm ranges while gaining the flexibility needed to handle regions with extreme GC content or complex secondary structures.
3Quantity of substance
If multiple heating/cooling cycles are used for probe hybridization, then complete probe binding is achieved, but process time increases and RNA degradation occurs
Solution Approach 1:
The patent applies preliminary action by designing probes with optimized parameters (melting temperatures, GC content, lengths) before hybridization. This pre-optimization ensures that probes are pre-configured to bind efficiently under simplified, single-step hybridization conditions, eliminating the need for multiple heating/cooling cycles and reducing both time and RNA degradation.
4Reliability
If probe sequences are extended to improve binding in difficult regions, then binding coverage is improved, but off-target matching increases
Solution Approach 1:
The patent changes probe parameters including length, GC content, and spacing to optimize binding coverage in difficult regions while controlling off-target effects. By carefully adjusting these parameters and using variable spacing rather than uniform tiling, the design achieves reliable binding without excessive sequence length that would increase off-target matching probability.
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 designed probes effectively remove superabundant RNAs, such as ribosomal and globin transcripts, enhancing the detection of rare mRNA transcripts, even in degraded samples, and reducing workflow time by half compared to conventional methods.
Implementation Method 1
hybridizing a plurality of DNA oligos to a target RNA molecule in a sample to form heteroduplexes
Implementation Method 2
digesting RNA in the heteroduplexes
Data Source
AI summary
The invention provides sets of RNA depletion probes, short DNA oligos that hybridize along the length of a target RNA and mediate digestion of the target RNA by RNase H to remove super-abundant RNA molecules from a sample. Depletion probes according to the invention are designed foremost based on biochemistry and the biophysical properties of the probes so that all of the depletion probes of a set exhibit substantially uniform, consistent behavior in binding to a target RNA in a sample. Probes are principally designed to specific performance targets and biophysical properties, yielding probe sets with irregular, even apparently random, spacing along a target RNA molecule.