Dataset Information

Characterisation of aptamer-anchored poly(EDMA-co-GMA) monolith for high throughput affinity binding.

ABSTRACT: Immobilisation of aptameric ligands on solid stationary supports for effective binding of target molecules requires understanding of the relationship between aptamer-polymer interactions and the conditions governing the mass transfer of the binding process. Herein, key process parameters affecting the molecular anchoring of a thrombin-binding aptamer (TBA) onto polymethacrylate monolith pore surface, and the binding characteristics of the resulting macroporous aptasensor were investigated. Molecular dynamics (MD) simulations of the TBA-thrombin binding indicated enhanced Guanine 4 (G4) structural stability of TBA upon interaction with thrombin in an ionic environment. Fourier-transform infrared spectroscopy and thermogravimetric analyses were used to characterise the available functional groups and thermo-molecular stability of the immobilised polymer generated with Schiff-base activation and immobilisation scheme. The initial degradation temperature of the polymethacrylate stationary support increased with each step of the Schiff-base process: poly(Ethylene glycol Dimethacrylate-co-Glycidyl methacrylate) or poly(EDMA-co-GMA) [196.0?°C (±1.8)]; poly(EDMA-co-GMA)-Ethylenediamine [235.9?°C (±6.1)]; poly(EDMA-co-GMA)-Ethylenediamine-Glutaraldehyde [255.4?°C (±2.7)]; and aptamer-modified monolith [273.7?°C (±2.5)]. These initial temperature increments reflected in the associated endothermic energies were determined with differential scanning calorimetry. The aptameric ligand density obtained after immobilisation was 480 pmol/?L. Increase in pH and ionic concentration affected the surface charge distribution and the binding characteristics of the aptamer-modified disk-monoliths, resulting in the optimum binding pH and ionic concentration of 8.0 and 5?mM Mg2+, respectively. These results are critical in understanding and setting parametric constraints indispensable to develop and enhance the performance of aptasensors.

SUBMITTER: Acquah C

PROVIDER: S-EPMC6787036 | biostudies-literature | 2019 Oct

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Characterisation of aptamer-anchored poly(EDMA-co-GMA) monolith for high throughput affinity binding.

Acquah Caleb C Chan Yi Wei YW Pan Sharadwata S Yon Lau Sie LS Ongkudon Clarence M CM Guo Haobo H Danquah Michael K MK

Scientific reports 20191010 1

Immobilisation of aptameric ligands on solid stationary supports for effective binding of target molecules requires understanding of the relationship between aptamer-polymer interactions and the conditions governing the mass transfer of the binding process. Herein, key process parameters affecting the molecular anchoring of a thrombin-binding aptamer (TBA) onto polymethacrylate monolith pore surface, and the binding characteristics of the resulting macroporous aptasensor were investigated. Molec ...[more]

PMID: 31601836

Similar Datasets

Project description:The high-resolution crystal structure of HIV-1 reverse transcriptase (RT) bound to a 38-mer DNA hairpin aptamer with low pM affinity was previously described. The high-affinity binding aptamer contained 2'-O-methyl modifications and a seven base-pair GC-rich tract and the structure of the RT-aptamer complex revealed specific contacts between RT and the template strand of the aptamer. Similar to all crystal structures of RT bound to nucleic acid template-primers, the aptamer bound RT with a bend in the duplex DNA. To understand the structural basis for the ultra-high-affinity aptamer binding, an integrative structural biology approach was used. Hydrogen-deuterium exchange coupled to liquid chromatography-mass spectrometry (HDX-MS) was used to examine the structural dynamics of RT alone and in the presence of the DNA aptamer. RT was selectively labeled with 15N to unambiguously identify peptides from each subunit. HDX of unliganded RT shows a mostly stable core. The p66 fingers and thumb subdomains, and the RNase H domain are relatively dynamic. HDX indicates that both the aptamer and a scrambled version significantly stabilize regions of RT that are dynamic in the absence of DNA. No substantial differences in RT dynamics are observed between aptamer and scrambled aptamer binding, despite a large difference in binding affinity. Small-angle X-ray scattering and circular dichroism spectroscopy were used to investigate the aptamer conformation in solution and revealed a pre-bent DNA that possesses both A- and B-form helical character. Both the 2'-O-methyl modifications and the GC tract appear to contribute to an energetically favorable conformation for binding to RT that contributes to the aptamer's ultra-high affinity for RT. The X-ray structure of RT with an RNA/DNA version of the aptamer at 2.8 Å resolution revealed a potential role of the hairpin positioning in affinity. Together, the data suggest that both the 2'-O-methyl modifications and the GC tract contribute to an energetically favorable conformation for high-affinity binding to RT.

Dataset Information

Characterisation of aptamer-anchored poly(EDMA-co-GMA) monolith for high throughput affinity binding.

Publications

Characterisation of aptamer-anchored poly(EDMA-co-GMA) monolith for high throughput affinity binding.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets