Expanded Genetic Alphabet Using Unnatural Base Pairs In Vivo
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Solution Overview
Problem
The limited chemical and physical diversity of the natural genetic alphabet restricts the potential functions and applications of oligonucleotides, such as in vitro evolution and biotechnological applications.
Innovation Solution
Incorporation of unnatural bases into nucleic acids within semi-synthetic organisms, such as Escherichia coli, to create an expanded genetic alphabet, enabling the formation of unnatural base pairs and enhancing the stability and functionality of nucleic acids.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If only natural nucleotides (A, C, G, T/U) are used in oligonucleotides, then the oligonucleotides can be synthesized and amplified by polymerases, but the chemical and physical diversity is limited, restricting potential functions and applications
Solution Approach 1:
The patent introduces unnatural base pairs (such as d5SICS-dNaM, dTPT3-dNaM, and other modified bases) that change the chemical parameters of the nucleic acid system. These unnatural bases have different chemical structures, sizes, and bonding properties compared to natural nucleotides, thereby expanding the chemical diversity and functional capabilities of oligonucleotides while maintaining compatibility with polymerase-based synthesis and amplification systems
Solution Approach 2:
The patent creates composite nucleic acid systems by combining natural nucleotides (A, C, G, T) with unnatural nucleotides (such as d5SICS, dNaM, dTPT3) to form hybrid base pairs. This composite approach allows the system to retain the proven functionality of natural nucleic acids while incorporating the enhanced chemical diversity and stability provided by unnatural components, enabling expanded applications in biotechnology and medicine
2Reliability
If unnatural bases are incorporated into nucleic acids, then the chemical diversity and stability are improved, but the complexity of the system increases
Solution Approach 1:
The patent applies local quality by incorporating unnatural base pairs at specific positions within oligonucleotides rather than uniformly replacing all natural bases. This allows the unnatural bases to provide localized stability enhancements and functional properties (such as increased resistance to nucleases or improved binding affinity) while the rest of the oligonucleotide maintains its natural structure and compatibility with biological systems
Solution Approach 2:
The unnatural base pairs introduced in the patent (such as d5SICS-dNaM and dTPT3-dNaM) possess altered physical and chemical parameters including increased thermal stability, enhanced resistance to enzymatic degradation, and modified binding characteristics. These parameter changes improve the reliability and stability of nucleic acid structures without requiring complete system redesign, as the unnatural bases are engineered to be compatible with existing polymerase mechanisms
Data Source
AI summary
Disclosed herein are in vivo methods, compositions, and kits for producing nucleic acids which comprises at least one unnatural base. Disclosed herein are in vivo methods of producing a nucleic acid with an expanded genetic alphabet, the method comprising incorporating at least one unnatural base in the nucleic acid. Disclosed herein are semi-synthetic organisms comprising an expanded genetic alphabet, wherein the genetic alphabet comprises at least one unnatural base. Disclosed herein are compositions that include a heterologous or recombinant polymerase and methods of use thereof. Further, disclosed herein are kits that are useful for stably incorporating an unnatural nucleic acid into a nucleic acid molecule, e.g., using the methods provided by the present invention in in vitro condition or under a cell free condition.