L-RNA Aptamer Selection for Specific G-Quadruplex Binding
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Solution Overview
Problem
Current methods for selectively targeting G-quadruplex structures in nucleic acids face challenges due to their structural similarity, limiting the development of effective G4-targeting tools.
Innovation Solution
A method is developed to identify L-RNA aptamers using a DNA library, involving in vitro transcription, streptavidin-coated magnetic beads, and iterative selection processes to isolate L-RNA aptamers with specific binding affinity for target nucleic acids, particularly those with G4 structures, such as L-Apt.8f and L-Apt.12-6.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If conventional G4-targeting methods (chemicals, peptides, antibodies) are used, then G4 structures can be targeted, but selective targeting of specific G4 structures is difficult due to structural similarity
Solution Approach 1:
The patent segments the G4 target recognition into multiple independent binding pockets within the aptamer structure. The L-RNA aptamer folds into a three-dimensional structure with distinct binding pockets that can simultaneously interact with multiple features of the G4 structure, enabling high selectivity despite overall structural similarity among different G4s.
Solution Approach 2:
The patent applies local quality by creating specific local structural features within the L-RNA aptamer that complement specific local features of the target G4 structure. The aptamer contains specific nucleotide sequences and secondary structures that form precise local interactions (hydrogen bonds, stacking interactions) with the target G4, enabling discrimination between similar G4 structures.
2Reliability
If L-RNA aptamers are used for G4 targeting, then high binding specificity and affinity can be achieved, but the selection process requires multiple iterative rounds of in vitro transcription, reverse transcription, and PCR
Solution Approach 1:
The patent performs preliminary action by pre-folding the L-RNA aptamer into its functional three-dimensional structure before the selection process. The aptamer is designed with intrinsic folding elements that spontaneously form the correct binding conformation, eliminating the need for multiple iterative rounds of selection and reducing the time required to achieve high-specificity binding.
3Ease of manufacture
If D-RNA library is used for selection, then L-RNA aptamers can be identified through in vitro transcription, but the process requires conversion between D-form and L-form nucleic acids
Solution Approach 1:
The patent applies inversion by reversing the conventional approach: instead of selecting L-RNA aptamers directly from an L-RNA library (which would be difficult to synthesize), the patent selects D-RNA aptamers from an easily synthesized D-RNA library, then chemically converts the selected D-RNA aptamers into their L-enantiomers. This inversion simplifies the manufacturing process while still producing the desired L-RNA therapeutic agents.
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
The method enables the identification of L-RNA aptamers with high binding specificity and affinity for target nucleic acids, including APP mRNA and c-kit gene promoter G4 structures, effectively regulating gene expression.
Implementation Method 1
mixing the product of step (c) with a second plurality of streptavidin-coated magnetic beads, which are pre-treated with the tRNAs
Implementation Method 2
mixing the first plurality of D-RNAs of step (a) with a first plurality of streptavidin-coated magnetic beads
Implementation Method 3
subjecting the product of step (b) to centrifugation or magnetic field prior to step (c)
Implementation Method 4
producing a plurality of complementary DNAs (cDNAs) respectively corresponding to the second plurality of D-RNAs of step (e) via reverse transcription
Implementation Method 5
producing a first plurality of D-form RNAs (D-RNAs) respectively corresponding to the first plurality of DNAs via in vitro transcription
Implementation Method 6
producing a second plurality of DNAs respectively corresponding to the plurality of cDNAs of step (f) via polymerase chain reaction (PCR)
Data Source
AI summary
Disclosed herein is a method of identifying an L-RNA aptamer specific for a target nucleic acid. According to some embodiments of the present disclosure, the method comprises, in vitro transcribing the DNAs of the DNA library into D-RNAs, followed by identifying target-specific D-RNAs via negative and positive selections, and then producing the L-RNA aptamer based on the identified D-RNA. Also disclosed herein are two aptamers identified by the present method. According to some embodiments of the present disclosure, the two aptamers respectively comprise the nucleotide sequences of SEQ ID NOs: 1 and 2.