G4-Specific Peptides for RNA G-Quadruplex Targeting
Solution Overview
Problem
Current methods for targeting G-quadruplex structures, such as G4-specific chemicals and antibodies, lack specificity and affinity for differentiating between RNA G4s and DNA G4s, and there is a need for innovative tools to recognize and regulate G4-mediated gene activity effectively.
Innovation Solution
A method involving in vitro transcription, puromycin attachment, translation, reverse transcription, and PCR is used to identify and select peptides specific to G4 structures from a DNA library, resulting in the identification of peptides like pep11, cyclic pep11, and tandem pep11, which exhibit binding affinity and specificity to target nucleic acids with G4 structures, enabling regulation of G4-mediated gene activity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If G4-specific chemicals and antibodies are used for targeting, then affinity for G4 structures is achieved, but specificity for differentiating between RNA G4s and DNA G4s deteriorates
Solution Approach 1:
The invention changes the chemical nature of the targeting molecule from small molecules/antibodies to peptides with specific amino acid compositions and structures. The identified peptides contain specific motifs (e.g., aromatic residues, basic residues) that enable selective recognition of rG4 structures through pi-stacking and electrostatic interactions, achieving both high affinity and RNA-specificity
Solution Approach 2:
The invention uses phage display technology to create a library of peptide variants that mimic natural G4-binding sequences. By displaying randomized peptide sequences on phage surfaces and selecting those that specifically bind rG4 structures, the method identifies peptide copies with optimized binding properties that differentiate RNA G4s from DNA G4s
2Reliability
If endogenous protein-derived peptides (RHAU23, RHAU53, RGG motif) are used, then G4-binding capability is achieved, but ability to regulate rG4-mediated gene activity deteriorates
Solution Approach 1:
The invention segments the G4-binding function from complex endogenous proteins into smaller, isolated peptide motifs. By identifying and isolating specific peptide sequences (e.g., 10-20 amino acid residues) that contain the essential G4-binding capability, the invention creates minimal peptides that can be optimized for regulatory functions without the interference of other protein domains
Solution Approach 2:
The invention creates a library of diverse peptide variants with varying sequences, lengths, and structures that can dynamically adapt to different rG4 targets and regulatory contexts. The phage display selection process identifies peptides with optimal dynamics for both binding and gene regulation functions
3Adaptability or versatility
If de novo peptides and nanobodies targeting dG4s are developed using phage display, then novel G4-targeting tools are created, but selectivity and affinity for rG4s deteriorates
Solution Approach 1:
Instead of selecting peptides that bind dG4 structures and hoping they will also bind rG4s, the invention inverts the selection process by specifically selecting for rG4 binding. The phage display library is screened against rG4 structures, and only peptides that demonstrate specific binding to RNA G4s are identified, ensuring inherent selectivity for the RNA substrate
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 identified peptides effectively bind to G4 structures, specifically targeting and regulating G4-mediated gene activity, providing a potential therapeutic approach for telomerase-related diseases like cancer and aging.
Implementation Method 1
producing a plurality of RNAs respectively corresponding to the first plurality of DNAs via in vitro transcription
Implementation Method 2
subjecting the product of step (b) to in vitro translation to produce a plurality of RNA-peptide conjugates
Implementation Method 3
subjecting the product of step (c) to reverse transcription to produce a first plurality of RNA-complementary DNA (cDNA)-peptide conjugates
Implementation Method 4
subjecting the product of step (f) to polymerase chain reaction (PCR) to produce a second plurality of DNAs
Implementation Method 5
attaching a puromycin molecule to the 3′ end of each of the plurality of RNAs of step (a) via a linker
Data Source
AI summary
Disclosed herein is a method of identifying a peptide specific to a target nucleic acid from a DNA library. According to some embodiments of the present disclosure, the method comprises, in vitro transcribing the DNAs of the DNA library into RNAs, producing RNA-cDNA-peptide conjugates from the RNAs by using a puromycin linker, selecting cDNAs from the RNA-cDNA-peptide conjugates exhibiting binding affinity to the target nucleic acid, and then identifying the peptide based on the selected cDNA. Also disclosed herein are the peptide identified by the present method, and uses thereof in treating telomerase-related diseases.