Springer Nature is developing a new tool to find and evaluate Protocols. Learn more. We are grateful to David Bishop for preparing, proofreading, and editing the final version of the manuscript and figures. Skip to main content. This service is more advanced with JavaScript available. These 3D DNA nanostructures can bind with hemin and significantly improve the intrinsic peroxidase activity of hemin.
Besides this function, they also enhance the fluorescence intensity of some G-quadruplex-specific dyes. Owing to these features, G-quadruplexes possess several superiorities in the detection of enzymes involved in nucleic acid metabolism, including facile probe fabrication without labeling, simple detection process without washing or separation steps, rapid observation by naked eyes, and easy integration with nucleic acid amplification strategies to amplify signals.
Herein, we describe two strategies for label-free detection of enzyme activity based on DNA G-quadruplexes. To increase sensitivity, template-dependent and template-independent DNA amplifications were introduced for the amplification of G-rich DNA sequences.
DNA methyltransferase and terminal deoxynucleotidyl transferase were detected as two model analytes, respectively. Springer Nature is developing a new tool to find and evaluate Protocols. Learn more. This timely publication is the first to offer practical application and direct strategies to meet the needs of emerging research.
Since there is usually only one specific DNA copy for regulatory purpose at a particular position in the genome, whereas house-keeping RNA and proteins are expressed at a low level with a few copies, the behaviors of biomolecules are stochastic.
For example, they undergo stochastic conformation changes in a living cell, which maintains numerous essential processes for life. To reveal the structural dynamics, heat and chemicals urea for instance have been conventionally serving as denaturants to trigger the conformation transitions between the native and denatured states in an ensemble manner. However, in a living cell, temperature is maintained constant, while chemical denaturants cannot reach to the concentration required to denature a biomolecule.
Instead, cells use mechanical force to induce the conformation transition for biomolecules, i. Optical tweezers serve as a standard tool to exert pico-Newton forces, which match with those involved in most force-based molecular events in a living cell. The prevalent occurrence of the G-quadruplex structures throughout the genome has made these structures hot targets for gene regulatory and pharmacological studies.
This thesis describes the characterization of three different human G-quadruplex DNA structures using single-molecule optical tweezers from a perspective of mechanical unfolding experiments. Introduction chapter provides a background on the biological relevance of G-quadruplex DNA and current available technology for G-quadruplex study.
Chapter IV is the most recent work on human telomeric G-quadruplex structure, which demonstrated how to apply optical tweezers to extract spatial information in a 3D fashion.
The last Chapter remarks the current work on G-quadruplexes and raises questions for future. The unique G-quadruplex secondary structures formed from guanine-rich DNA are implicated as anti-cancer targets for their ability to inhibit overactive DNA polymerase and aberrant gene expression. As the body's natural on-off switch for controlling cell life cycles, stabilisation of G-quadruplex by external ligands can reduce cellular dysfunction. Despite the extensive research on G-quadruplex stabilising agents, few ligands have reached clinical trials and moved further onto the market.
Selectivity and potency issues can hinder this process. Through rational design, we developed three series of ligands to enhance potency, selectivity, and cell permeability: two aromatic planar series targeting G-tetrads, and one groove-binding series targeting a c-kit G-quadruplex groove site. For the first series, we used Schrodinger's Glide molecular docking software and an arbitrary selection of ligands from an extensive ligand database to perform a virtual screen on the ckit1 groove site.
Results showed triangular ligands such as triarylpyridines and triarylpyrimidines were potent groove-binding ligands. From these docking results, we synthesised two 4'-substituted 2,,2"-terpyridine derivatives and purchased three 2,,2"-terpyridine ligands with reported low G-quadruplex selectivity as negative controlsfor surface plasmon resonance SPR analysis. In the second series, the parallel synthesis of linear, crescent, and triangular-shaped arylcarboxamides through amide coupling allowed the development of an extensive ligand library to ascertain the best pharmacophore for specific G-quadruplex structures.
Initial attempts to synthesise a crescent 1- 4-aminophenyl guanidine derivative was successful, however the method used was not transferrable to the linear and triangular counterparts. Following an alternate literature method, aniline and 3-aminopyridine derivatives were synthesised through isolation from a reagent, sym-collidine, appeared difficult.
Using this method the linear 1- 4-aminophenyl guanidine derivative was synthesised, though onlyintermediates were isolated for triangular derivative. The third series, we postulated triazolophane macrocycles to bind to G-tetrad surface due to its high planarity and aromaticity.
A rationally designed triazolophane was devised with a pyridyl-based core featuring propylguanidine groups through ester and ether appendages for enhanced binding with the DNA backbone. The multistep synthesis of phenyl diazide and pyridyl dialkyne building blocks has thus far been fruitless as ideal "click" conditions are yet to be discovered for full triazolophane synthesis. Finally, SPR analysis of a model triazolophane, the crescent guanidine-basedarylcarboxamide, and the five terpyridine ligands provided the impetus for further G-quadruplex ligand development.
The terpyridine series showed no selectivity for cki1 and its groove site, or preference for G-quadruplex DNA - alternative molecular docking programs are required to determine an ideal ckitl-specific groove binding agent.
Both the arylcarboxamide and model triazolophane displayed G-quadruplex selective binding - these positive results supports the full development of our arylcarboxamide ligand database and further analysis for the development of triazolophane macrocycles.
While further research is needed to complete our goals, these intitial binding studies reveal two novel classes of anti-cancer agents targeting G-quadruplex DNA. In vitro, G-rich sequences form highly stable secondary structures known as G-Quadruplexes.
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