3′-O-Glycoside-Blocked Nucleotides for Enzymatic DNA Sequencing
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Solution Overview
Problem
Existing sequencing technologies face challenges in efficiently incorporating nucleotides while preventing unwanted secondary incorporations, requiring blocking groups that are stable yet easily removable without damaging the DNA structure, and current methods often involve costly and potentially damaging chemical catalysts.
Innovation Solution
Development of nucleotides with enzymatically removable 3′-O-glycoside blocking groups that allow for precise nucleotide incorporation and subsequent enzymatic cleavage, using recombinant enzymes for stability and cost-effectiveness.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If chemical catalysts are used to remove blocking groups, then nucleotide incorporation can proceed, but DNA damage and signal decay increase
Solution Approach 1:
The patent replaces chemical catalysts with enzymatic systems for blocking group removal. Specifically, it uses recombinant glycosidases and phosphatases to cleave 3'-O-glycoside blocking groups instead of chemical catalysts, thereby reducing DNA damage and signal decay while maintaining nucleotide incorporation efficiency
Solution Approach 2:
The patent changes the chemical nature of the blocking group from traditional groups requiring harsh chemical removal to 3'-O-glycoside groups that can be selectively removed by enzymatic hydrolysis. This parameter change allows for milder, more DNA-friendly deprotection conditions
2Reliability
If traditional blocking groups are used, then nucleotide incorporation is controlled, but removal conditions damage the DNA structure
Solution Approach 1:
The patent introduces enzymatic systems as intermediaries between the blocked nucleotide and the DNA chain. Recombinant glycosidases and phosphatases act as mediators to selectively remove blocking groups under mild conditions, preserving DNA structure integrity while maintaining incorporation control
Solution Approach 2:
The patent employs disposable blocking groups (3'-O-glycosides) that are designed for single-use functionality. These blocking groups are easily removed by inexpensive recombinant enzymes after serving their purpose of preventing unwanted secondary incorporations, eliminating the need for costly or damaging removal procedures
3Ease of operation
If chemical deprotection methods are used, then blocking groups are removed, but production costs increase
Solution Approach 1:
The patent employs self-service deprotection where recombinant enzymes specifically recognize and remove their corresponding blocking groups. The enzymatic systems are designed to be highly specific, automatically removing only the intended blocking groups without requiring complex chemical protocols, thereby reducing production costs
Solution Approach 2:
The patent changes the deprotection mechanism from chemical to enzymatic, enabling milder reaction conditions and reducing production costs. The use of recombinant enzymes allows for cost-effective large-scale production and simplifies the deprotection process
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
Enzymatic cleavage of 3′-O-glycoside blocking groups provides less DNA damage and improved stability during storage, reducing signal decay and phasing errors in sequencing-by-synthesis methods, while enabling cost-effective large-scale enzyme production.
Implementation Method 1
enzymatically removable 3′ blocking group in the form of a 3′-O-glycoside group
Implementation Method 2
using recombinant enzymes for stability and cost-effectiveness
Data Source
AI summary
Embodiments of the present disclosure relate to nucleotide and nucleoside molecules with 3′-O-glycoside blocking groups. Also provided herein are methods to prepare such nucleotide and nucleoside molecules, and methods and kits for sequencing applications.