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Evidence of protein-free homology recognition in magnetic bead force-extension experiments.

ABSTRACT: Earlier theoretical studies have proposed that the homology-dependent pairing of large tracts of dsDNA may be due to physical interactions between homologous regions. Such interactions could contribute to the sequence-dependent pairing of chromosome regions that may occur in the presence or the absence of double-strand breaks. Several experiments have indicated the recognition of homologous sequences in pure electrolytic solutions without proteins. Here, we report single-molecule force experiments with a designed 60?kb long dsDNA construct; one end attached to a solid surface and the other end to a magnetic bead. The 60?kb constructs contain two 10?kb long homologous tracts oriented head to head, so that their sequences match if the two tracts fold on each other. The distance between the bead and the surface is measured as a function of the force applied to the bead. At low forces, the construct molecules extend substantially less than normal, control dsDNA, indicating the existence of preferential interaction between the homologous regions. The force increase causes no abrupt but continuous unfolding of the paired homologous regions. Simple semi-phenomenological models of the unfolding mechanics are proposed, and their predictions are compared with the data.

SUBMITTER: O' Lee DJ

PROVIDER: S-EPMC4971244 | biostudies-literature | 2016 Jul

REPOSITORIES: biostudies-literature

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Evidence of protein-free homology recognition in magnetic bead force-extension experiments.

O' Lee D J DJ Danilowicz C C Rochester C C Kornyshev A A AA Prentiss M M

Proceedings. Mathematical, physical, and engineering sciences 20160701 2191

Earlier theoretical studies have proposed that the homology-dependent pairing of large tracts of dsDNA may be due to physical interactions between homologous regions. Such interactions could contribute to the sequence-dependent pairing of chromosome regions that may occur in the presence or the absence of double-strand breaks. Several experiments have indicated the recognition of homologous sequences in pure electrolytic solutions without proteins. Here, we report single-molecule force experimen ...[more]

PMID: 27493568

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Project description:Proteins lack the duplication mechanism like nucleic acids, so the connection of immunoassays with effective nucleic acid amplification techniques has become a powerful way for the detection of trace protein biomarkers in biological fluids. However, such immunoassays generally suffer from rather stringent DNA sequence design and complicated operations. Herein, we propose a simple but highly sensitive flow cytometric immunoassay (FCI) by employing on-bead terminal deoxynucleotidyl transferase (TdT)-initiated template-free DNA extension as an effective signal amplification pathway (TdT-FCI), and gold nanoparticles (AuNPs) co-functionalized with both the detection antibody and a 3'-OH oligonucleotide (ODN) as the transducer to bridge the immunoassay and subsequent TdT-mediated DNA amplification. The target antigen can sandwich with the capture antibody immobilized on the magnetic beads (MBs) and the detection antibody on the AuNPs to bring a lot of ODNs onto the surface of MBs. Each ODN on the MBs can be effectively elongated by TdT in a template-free manner to produce a long poly(T) tail, which will then bind to many 6-carboxyfluorescein (FAM)-labeled poly(A)25. Since each AuNP can carry multiple ODNs and each extended ODN can ultimately capture numerous FAM-poly(A)25, efficiently amplified fluorophore accumulation on the MBs can be achieved. The fluorescent MBs can be individually interrogated with a flow cytometer and thus quantitative analysis of the target antigen can be realized. Coupled with the powerful flow cytometry analysis, the simple but efficient TdT-based signal amplification mechanism has pushed the detection limit of prostate specific antigen (PSA) down to a low level of 0.5 pg mL-1. Furthermore, based on an elegant bead size-encoding principle, we have further advanced the TdT-FCI for multiplexed antigen detection in a single reaction. Sharing the unique merits of simple design and operation, efficient signal amplification, powerful signal readout and the capability for multiplexed analysis, this TdT-FCI provides a versatile tool for detecting trace antigen biomarkers towards clinical diagnosis as well as prognosis.

Dataset Information

Evidence of protein-free homology recognition in magnetic bead force-extension experiments.

Publications

Evidence of protein-free homology recognition in magnetic bead force-extension experiments.

Similar Datasets

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