Chemical Sequencing of L-Nucleic Acids via Cleavage
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Solution Overview
Problem
Current methods lack a sensitive, accurate, and reproducible technique for sequencing mirror-image nucleic acids, particularly L-nucleic acids, due to the absence of practical methods for incorporating labeled L-di-deoxynucleotide triphosphates or L-deoxyribonucleotide triphosphates and the unavailability of specific D-nucleic acid polymerases or helicases required for sequencing-by-synthesis and nanopore technologies.
Innovation Solution
A chemical sequencing method using chemicals like Dimethyl sulfate, Methylamine, Methylene blue, Sodium hydroxide, and Hydrazine hydrate, combined with gel-electrophoresis and labeling with 5-iodoacetamidofluorescein, to determine the sequence of L-nucleic acid sequences, which can also involve reverse transcription of ribose nucleic acids into deoxyribose nucleic acids using Sulfolobus solfataricus P2 DNA polymerase IV (Dpo4) for further analysis.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If sequencing-by-synthesis methods are used, then sequencing capability is improved, but the method is unavailable because no polymerase can incorporate labeled L-ddNTPs or L-dNTPs
Solution Approach 1:
The patent replaces enzymatic sequencing methods with chemical sequencing methods. Specifically, it uses chemical cleavage reagents (such as hydroxylamine for cytosine, formic acid for purines, and other base-specific chemicals) to selectively cleave L-nucleic acid sequences at different nucleotide positions, enabling sequencing without requiring L-polymerases or L-nucleotides.
Solution Approach 2:
The patent introduces chemical reagents as intermediaries to achieve sequencing. These reagents (hydroxylamine, formic acid, dimethyl sulfate, etc.) act as mediators that selectively modify or cleave specific bases in L-nucleic acids, allowing sequence determination through detection of cleavage patterns without direct enzymatic involvement.
2Measurement precision
If nanopore sequencing is used, then sequencing capability is improved, but it requires unavailable D-nucleic acid polymerase or helicase
Solution Approach 1:
The patent replaces the mechanical/enzymatic nanopore sequencing approach with a chemical sequencing system. Instead of using nanopores that require D-polymerases or D-helicases to manipulate L-DNA, the invention uses chemical reagents that directly interact with L-nucleic acids to produce detectable cleavage patterns, eliminating the need for unavailable enzymatic components.
Solution Approach 2:
The patent adapts established chemical sequencing methodologies (originally developed for D-nucleic acids) and applies them to L-nucleic acids. By copying the chemical principles from D-nucleic acid sequencing and demonstrating their applicability to L-nucleic acids, the invention makes sequencing feasible without requiring new enzymatic systems.
3Adaptability or versatility
If chemical sequencing method is used, then sequencing of L-nucleic acids is enabled, but labeling and detection sensitivity must be improved
Solution Approach 1:
The patent employs fluorescent labeling of L-nucleic acids and detects sequence information through fluorescence signals. The chemical cleavage reagents produce fragments that are detected via their fluorescent labels, with the fluorescence intensity and patterns providing sensitive detection of the sequence. This optical detection method significantly improves sensitivity compared to non-fluorescent methods.
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 accurate sequencing of L-nucleic acid sequences, including those over 120 nucleotides in length, and facilitates applications such as amplification, cloning, and transcriptome analysis by generating labeled fragments that can be analyzed for sequence determination.
Implementation Method 1
the chemical sequencing method comprises gel-electrophoresis to determine the nucleic acid sequence
Implementation Method 2
labeling at a 5′ terminus or 3′ terminus of the nucleic acid sequence comprising the L-nucleotides with 5-iodoacetamidofluorescein
Implementation Method 3
subjecting the nucleic acid sequence comprising the L-nucleotides to a chemical sequencing method using a chemical selected from the group consisting of Dimethyl sulfate, Methylamine, Diethyl pyrocarbonate, Methylene blue, Potassium chloropalladate, Sodium hydroxide, Osmium tetroxide, Spermine, potassium permanganate, Hydrazine, hydrazine hydrate, Hydroxylamine hydrochloride, Diethyl pyrocarbonate, Formic acid and Citrate buffer
Implementation Method 4
reverse transcription of ribose nucleic acids into deoxyribose nucleic acids using Sulfolobus solfataricus P2 DNA polymerase IV (Dpo4)
Data Source
AI summary
Methods of sequencing and producing nucleic acid sequences are provided. Accordingly there are provided methods of sequencing a nucleic acid sequence comprising L-nucleotides comprising subjecting the nucleic acid sequence comprising the L-nucleotides to a chemical sequencing method. Also provided is a method of reverse transcribing a ribose nucleic acid sequence into a deoxyribose nucleic acid sequence comprising catalyzing reverse transcription of the ribose nucleic acid sequence with a Sulfolobus solfataricus P2 DNA polymerase IV (Dpo4).