Segmented Nucleic Acids via Non-Nucleotide Linkers
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Solution Overview
Problem
The synthesis of long nucleic acids with two or more non-nucleotide linkers is inefficient, leading to challenges in manufacturing, purification, and analytical characterization, and existing methods result in high synthetic errors and off-target cleavage in gene editing applications.
Innovation Solution
A highly efficient chemical method for preparing segmented nucleic acids using non-nucleotide linkers, which are formed by chemical ligations and enhance the function of nucleic acids by altering secondary structures and introducing additional molecular interactions, including hydrogen bonds, and are used in conjunction with CRISPR-mediated gene editing.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If conventional synthesis methods are used for long nucleic acids with multiple non-nucleotide linkers, then the synthesis can be performed with existing techniques, but the synthesis efficiency is low and synthetic errors are high
Solution Approach 1:
The nucleic acid is divided into multiple segments that are synthesized separately and then joined together through chemical ligation. This segmentation approach allows each segment to be synthesized with high precision using conventional methods, while the overall assembly achieves high efficiency through parallel processing and modular construction, resolving the contradiction between synthesis efficiency and manufacturing precision.
2Ease of manufacture
If non-nucleotide linkers are introduced to replace phosphate diester bonds, then manufacturing cost is reduced and chemical modifications are enabled, but synthesis methods become more complex and challenging
Solution Approach 1:
Chemical linkers serve as intermediary components that connect nucleic acid segments through well-established chemical ligation reactions. These linkers provide standardized interfaces with defined chemistry, enabling modular assembly while maintaining synthetic tractability. The intermediary nature of the linkers allows for systematic optimization of synthesis protocols, reducing overall complexity despite the introduction of non-standard components.
3Device complexity
If long nucleic acids are synthesized as single continuous strands, then the structure is simple, but purification and analytical characterization become challenging due to secondary structures
Solution Approach 1:
By synthesizing the nucleic acid as separate segments that are subsequently ligated, the method enables purification of individual segments before assembly. This segmentation strategy simplifies purification and analytical characterization at each stage, as shorter segments form fewer complex secondary structures compared to the full-length continuous strand. The final assembled structure achieves the desired functionality while maintaining synthetic and analytical tractability.
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 cost-effective, high-quality production of segmented nucleic acids with reduced synthetic errors, improved stability, and targeted delivery, enhancing the efficacy and selectivity of gene editing therapies.
Implementation Method 1
non-nucleotide linkers formed by chemical ligations
Implementation Method 2
introducing additional molecular interactions including hydrogen bonds
Data Source
AI summary
Provided herein are processes and methods for preparation of segmented nucleic acids and segmented nucleic acid conjugates comprising at least two non-nucleotide linkers, and their RNP complexes with RNA guided gene editing proteins including CRISPR Cas proteins and ADAR enzymes. Also disclosed are the uses of the compositions comprising segmented nucleic acids or segmented nucleic acid conjugates as medicinal agents for treatment of diseases.