L-AEGIS Oligonucleotides for Nuclease-Resistant Aptamer Stability
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Solution Overview
Problem
Existing methods for in vitro selection of nucleic acid molecules, such as SELEX, face challenges in maintaining information density and stability of aptamers and aptazymes during PCR amplification and in biological media, particularly with enzymes that digest standard xNA molecules but not those with L-configuration.
Innovation Solution
Development of artificially expanded genetic information system (AEGIS) oligonucleotides using non-standard nucleobases attached to L-nucleosides, which are stable in biological environments and can be amplified using rolling circle amplification, and the use of L-AEGIS oligonucleotides that mimic D-AEGIS binding properties.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If standard xNA molecules are used in in vitro selection processes, then PCR amplification can be performed efficiently, but the molecules are digested by nucleases in biological media leading to loss of stability
Solution Approach 1:
The patent applies L-nucleosides (mirror image of standard D-nucleosides) to create L-xNA molecules that are resistant to natural nucleases. By inverting the stereochemical configuration, the molecules gain stability in biological media while maintaining the ability to undergo PCR amplification and form functional aptamers.
Solution Approach 2:
The patent changes the stereochemical parameter of the nucleoside configuration from D to L, which fundamentally alters the molecule's interaction with biological enzymes. This parameter change confers nuclease resistance while preserving the essential properties needed for in vitro selection and amplification processes.
2Reliability
If L-configuration nucleosides are used to resist nuclease digestion, then stability in biological media is improved, but standard PCR amplification processes cannot be used
Solution Approach 1:
By using L-nucleosides instead of D-nucleosides, the patent creates molecules that evade nuclease digestion. The inverted configuration allows these L-xNA molecules to be amplified using modified PCR protocols and to form functional aptamers, thus maintaining productivity while gaining stability.
3Adaptability or versatility
If information density is increased in aptamers, then binding efficiency is improved, but the complexity of selection and amplification processes increases
Solution Approach 1:
The patent increases information density by incorporating non-standard nucleobases with different hydrogen bonding patterns into the L-xNA molecules. This parameter change enables more complex binding specificities and higher affinity interactions while the standardized L-nucleoside backbone maintains compatibility with established amplification methodologies.
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
Enhances the stability and binding efficiency of aptamers and aptazymes in complex biological media and maintains high information density by using L-AEGIS oligonucleotides that are resistant to nucleases and can replicate effectively through RCA, overcoming the limitations of standard PCR processes.
Implementation Method 1
L-AEGIS oligonucleotides that are resistant to nucleases
Implementation Method 2
can be amplified using rolling circle amplification
Implementation Method 3
present, to a complementary strand in a Watson-Crick pairing geometry, a pattern of hydrogen bonds that is different from the pattern presented by adenine, guanine, cytosine, and uracil
Data Source
AI summary
This invention provides for processes for creating a long DNA molecule that is a concatamer that comprises a repeating oligonucleotide segment, wherein one or more of the nucleotides of said segment has one or more independently selected templating “non-standard” nucleotides. These are nucleotide analogs that, when incorporated into oligonucleotides (DNA or RNA, collectively xNA), present to a pattern of hydrogen bonds that is different from the pattern presented by adenine, guanine, cytosine, and uracil. This disclosure provides a process for obtaining these using rolling circle amplification.