Triple-Stranded Nucleic Acid TFO Binding Affinity
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Solution Overview
Problem
Existing methods for improving the binding affinity of triplex forming oligonucleotides (TFO) to pyrimidine nucleotides in double-stranded DNA (dsDNA) have been unsuccessful due to modifications that adversely affect the stability and solubility of TFO.
Innovation Solution
The use of modified deoxyuridine triphosphates (dUTPs), such as 5-fluoro-uridine, 5-chloro-uridine, 5-bromo-uridine, and 5-formyl-uridine, in PCR reactions to produce double-stranded nucleic acids that enhance the binding affinity of TFO to dsDNA by forming a triple-stranded nucleic acid complex.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If TFO is modified to improve binding affinity to pyrimidine-containing strand, then binding affinity is improved, but stability and solubility of TFO deteriorate
Solution Approach 1:
The patent changes the chemical parameters of the nucleotides by incorporating modified bases (5-fluorocytosine, 5-chlorocytosine, 5-bromocytosine, 5-formylcytosine) instead of standard cytosine. These parameter changes in the chemical structure allow TFO to maintain high binding affinity to pyrimidine-containing strands while preserving stability and solubility properties.
2Reliability
If TFO binds to purine-rich strand, then binding affinity is maintained, but ability to bind to pyrimidine-containing strand deteriorates
Solution Approach 1:
The patent applies local quality by making the TFO sequence composition specific to the target region. The TFO is designed with a high proportion of modified cytosine analogs (at least 50% of nucleotides) that locally enhance binding capability to pyrimidine-containing strands through Hoogsteen or reverse Hoogsteen base pairing, while maintaining overall triplex structure stability.
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 improves the binding affinity of TFO to dsDNA, allowing for more effective detection of target sequences through the formation of stable triplex complexes, as demonstrated by the increased fluorescence signal in the presence of modified dUTPs.
Implementation Method 1
the first strand binds to the TFO via Hoogsteen base pairing or reverse Hoogsteen base pairing
Implementation Method 2
the first strand binds to the TFO via Hoogsteen base pairing or reverse Hoogsteen base pairing
Implementation Method 3
the second strand comprises a plurality of modified nucleotides... significantly improves the binding affinity of TFO to dsDNA
Data Source
AI summary
Disclosed herein is a triple-stranded nucleic acid comprising a double-stranded nucleic acid and a triplex forming oligonucleotide (TFO), in which the double-stranded nucleic acid comprises a first strand and a second strand complementary to the first strand, and the TFO binds to the first strand. According to some embodiments of the present disclosure, the second strand comprises a plurality of modified nucleotides independently selected from the group consisting of 5-fluoro-uridine, 5-chloro-uridine, 5-bromo-uridine and 5-formyl-uridine nucleotides. Also disclosed herein are kits and methods of detecting a target sequence in a double-stranded nucleic acid.