Modular Hybridization Probes for Long Sequence Capture
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Solution Overview
Problem
Current nucleic acid analysis techniques, such as PCR and next-generation sequencing, struggle with analyzing long or complex sequences, particularly trinucleotide repeats, due to limitations in hybridization probe length, specificity, and tolerance for sequence variations, which are essential for profiling diverse DNA samples like T-cell receptors and antibody fragments.
Innovation Solution
A modular nucleic acid probe design featuring complementary oligonucleotides that collectively span and hybridize to long target sequences, incorporating molecular competitor species for specificity, allowing for the detection and quantification of long target sequences and combinatorial diversity, overcoming limitations of previous probe designs.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Length of moving object
If standard hybridization probes are used, then synthesis is straightforward, but probe length is limited by synthesis capabilities and cannot query long target regions
Solution Approach 1:
The probe is divided into multiple modular segments (e.g., 50-150 nucleotides each) that can be synthesized separately using standard oligonucleotide synthesis capabilities. These segments are then assembled in vitro to form long probes (500-2000+ nucleotides) that can query extended target regions while maintaining ease of manufacture through modular construction.
2Adaptability or versatility
If standard hybridization probes are used, then they can bind to target sequences, but they are not economical for profiling DNA samples with combinatorial diversity
Solution Approach 1:
The probe design incorporates universal regions (e.g., fluorophore-labeled and quencher-containing segments) that can be combined with various target-specific segments. This modular universal design allows the same probe architecture to profile multiple different DNA samples with combinatorial diversity (such as T-cell receptors and antibody fragments) without requiring completely new probe designs for each application.
Solution Approach 2:
The probe is segmented into interchangeable modules including target-complementary segments and universal functional segments. This segmentation enables combinatorial assembly of probes tailored to different target sequences while maintaining consistent detection methodology, making the system economical for profiling diverse DNA samples.
3Measurement precision
If standard hybridization probes are used, then they can detect target sequences, but they are incapable of accurate quantitation of trinucleotide repeats
Solution Approach 1:
The probe design incorporates dynamic toehold-mediated strand displacement mechanisms that enable progressive hybridization along the target sequence. This dynamic process allows the probe to accurately sense and quantify trinucleotide repeat lengths (such as in Huntington's disease, Fragile X, and Friedreich's ataxia) by measuring the extent of hybridization, overcoming the static limitations of standard probes that cannot accurately measure repetitive sequences.
4Length of moving object
If modular probe design with complementary sections is used, then probe length can be extended, but specificity is reduced because once any section binds it recruits the other sections
Solution Approach 1:
The probe design incorporates toehold regions that initiate hybridization at specific locations on the target sequence before full hybridization occurs. This preliminary action ensures that binding occurs only at the correct target site with proper sequence complementarity, preventing false recruitment of probe sections to non-specific sites while still enabling extended probe length through modular segmentation.
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 specific detection and quantification of long target sequences, including triplet repeats, and supports combinatorial profiling applications, providing sequence variation tolerance and nucleotide selectivity, thereby improving analysis of complex nucleic acid sequences.
Implementation Method 1
Complement oligonucleotides that collectively span and hybridize to a long target sequence
Implementation Method 2
the modular probe also includes molecular competitor species with sequence similar to the target, to ensure hybridization specificity through molecular competition
Data Source
AI summary
This present disclosure describes hybridization probes modularly constructed from several oligonucleotides with a pattern of designed complementary interactions, allowing the probes to sequence-specifically capture or analyze nucleic acid target sequences that are long and/or complex.