Oligonucleotide Tagging Probes for MicroRNA Quantification
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Solution Overview
Problem
Current methods for detecting and quantifying microRNAs and siRNAs are limited by their small size and low expression levels, leading to low sensitivity and throughput, particularly in distinguishing between mature and precursor forms, which is crucial for understanding their role in human diseases like cancer.
Innovation Solution
The use of novel oligonucleotide tagging probes with high affinity nucleotide analogues, such as LNA, for specific hybridization and ligation, followed by real-time quantitative PCR, allows for accurate detection and quantification of short RNA sequences, including microRNAs and siRNAs, in complex nucleic acid samples.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional detection methods are used for microRNAs and siRNAs, then the detection process is simpler, but the sensitivity and measurement precision are insufficient due to the small size and low expression levels of these RNAs
Solution Approach 1:
The detection method is divided into multiple sequential steps: hybridization of tagging probes to target RNA, ligation of probes to form concatemers, reverse transcription to cDNA, and quantitative PCR amplification. This segmentation allows each step to be optimized independently, achieving high sensitivity for detecting low-abundance microRNAs and siRNAs while maintaining manageable procedural complexity through standardized protocols.
Solution Approach 2:
Tagging probes with high-affinity nucleotide analogues (LNA, 2'-O-methyl RNA) serve as intermediaries that specifically bind to target microRNAs and siRNAs. These probes act as mediators between the target RNA and the detection system, enabling sensitive detection of low-expression RNAs through their high binding affinity and specificity, thereby improving measurement precision without requiring direct complex interaction between detection reagents and target molecules.
2Measurement precision
If conventional methods are used, then the procedure is less complex, but the ability to distinguish between mature and precursor forms is insufficient
Solution Approach 1:
The tagging probes are designed with sequence specificity targeting distinct regions of mature microRNAs versus their precursor forms. By placing high-affinity nucleotide analogues at specific positions within the probe sequence, the method achieves local enhancement of binding discrimination, allowing clear distinction between mature and precursor forms based on their sequence differences while maintaining overall assay simplicity.
Solution Approach 2:
The method performs preliminary hybridization and ligation steps before amplification, where tagging probes specifically bind to and mark target molecules of interest. This preliminary action occurs under optimized conditions that favor specific binding, enabling subsequent discrimination between mature and precursor forms to be achieved with high accuracy without requiring complex analysis procedures later in the workflow.
3Reliability
If standard oligonucleotide probes are used, then the assay is easier to perform, but the hybridization affinity is insufficient for short RNA targets
Solution Approach 1:
The tagging probes are constructed as composite oligonucleotides incorporating high-affinity nucleotide analogues (LNA, 2'-O-methyl RNA) mixed with standard nucleotides. This composite structure combines the ease of standard oligonucleotide synthesis with the enhanced binding properties of modified nucleotides, achieving reliable hybridization to short RNA targets while maintaining practical probe manufacturing through commercially available synthesis capabilities.
Solution Approach 2:
The method changes the chemical parameters of the oligonucleotide probes by incorporating nucleotide analogues with higher binding affinity and thermal stability. This parameter change increases hybridization strength and specificity for short RNA targets without fundamentally altering the probe synthesis process, as the modified nucleotides can be incorporated using standard phosphoramidite chemistry with minor protocol adjustments.
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 enables sensitive and specific detection of microRNAs and siRNAs at low concentrations, distinguishing between mature and precursor forms, thereby improving our understanding of their expression and role in diseases, and facilitating their use in diagnostics and research.
Implementation Method 1
The first tagging probe hybridizes to a first region within a target sequence and the second tagging probe hybridizes to a second region within the same complementary target sequence
Implementation Method 2
The background in the hybridization to the target RNA sequence in complex nucleic acid samples is eliminated by the use of two tagging probes, where the hybridization of both probes to the complementary target sequence is required for the covalent joining of the two probes
Data Source
AI summary
The invention relates to ribonucleic acids, probes and methods for detection, quantification as well as monitoring the expression of mature microRNAs and small interfering RNAs (siRNAs). The invention furthermore relates to methods for monitoring the expression of other non-coding RNAs, mRNA splice variants, as well as detecting and quantifying RNA editing, allelic variants of single transcripts, mutations, deletions, or duplications of particular exons in transcripts, e.g., alterations associated with human disease such as cancer. The invention furthermore relates to methods for detection, quantification as well as monitoring the expression of deoxy nucleic acids.