Triplex-Forming Tag for Universal Protein Isolation
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Solution Overview
Problem
Current methods for isolating specific chromatin fragments associated with particular genes are inefficient due to the large size of genomes and the inability to isolate proteins bound to any nucleic acid sequence independently of its location or sequence, limiting understanding of gene expression and regulation.
Innovation Solution
The use of a specific double-stranded DNA sequence, known as a Triplex-Forming Tag (TFT) sequence, which forms a stable triple helix with a Triplex Forming Oligonucleotide (TFO) probe, allows for the isolation and identification of proteins bound to any nucleic acid sequence of interest by introducing the TFT sequence nearby the target sequence and using the TFO probe to purify the associated proteins.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If Watson-Crick hybridization is used to isolate chromatin fragments, then telomere fragments can be enriched, but the method is limited to telomeres only and cannot isolate fragments from any location in the genome
Solution Approach 1:
The patent introduces a triplex-forming tag (TFT) sequence as an intermediary element that can be inserted near any gene of interest. This TFT sequence serves as a universal mediator that allows the TFO probe to bind to chromatin fragments regardless of their original genomic location or sequence, thereby enabling versatile isolation while maintaining high efficiency through the stable triplex structure.
Solution Approach 2:
The patent changes the binding mechanism from Watson-Crick base pairing to triple helix formation. By using a TFO probe that forms a stable triplex structure with the TFT tag, the method achieves both high binding affinity (improving isolation efficiency) and universal applicability to any nucleic acid sequence containing the TFT tag).
2Quantity of substance
If chromatin isolation strategies are used to establish locus-specific protein composition, then enrichment of targeted region is achieved, but material in sufficient amount and purity is not obtained
Solution Approach 1:
The patent performs preliminary action by inserting the TFT sequence near the gene of interest before chromatin isolation. This pre-tagging approach ensures that when the TFO probe is applied, it can efficiently capture the target chromatin fragment along with its bound proteins, yielding sufficient material in high purity without requiring extensive post-isolation purification steps.
3Measurement precision
If one attempts to isolate chromatin fragments associated with a single human gene, then locus-specific protein composition can be studied, but the isolation of 1 out of 10^6 fragments is extremely demanding
Solution Approach 1:
The TFT sequence acts as a high-specificity intermediary marker that can be inserted near any gene of interest. This mediator enables the TFO probe to specifically recognize and bind to the target chromatin fragment among millions of other fragments, achieving both high specificity (1 out of 10^6 isolation accuracy) and high productivity through the stable and specific triplex interaction.
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 method enables the efficient isolation and analysis of proteins bound to specific nucleic acid sequences, overcoming the limitations of existing techniques by allowing for the purification of proteins from complex mixtures, regardless of the nucleic acid sequence or location, thereby facilitating the study of gene regulation and expression.
Implementation Method 1
the TFT sequence can form a stable complex in the form of a triple helix with a specific oligonucleotide probe referred to as the Triplex Forming Oligonucleotide (TFO)
Data Source
AI summary
The invention is to supply a novel way for the isolation and identification of proteins bound to any kind of interesting nucleic acid sequence (Sequence-of-Interest: SoI), advantageously to any kind of interesting DNA sequence, particularly in the context of chromosomal DNA or RNA or episomal DNA in living cells or in test tubes.In the context of the present invention, living cells include any organism that contains nucleic acid material as for example viruses, bacteria, cells, the bound protein of which have to be analyzed.The invention is based upon the use of a specific nucleic acid sequence tag, advantageously a specific double-stranded DNA able to form triplex helix, referred to as the Triplex-Forming Tag sequence, (TFT sequence), that will be located nearby the SoI.