Atomic Force Microscopy Methods for<scp>DNA</scp>Analysis

Lyubchenko, Yuri L.

doi:10.1002/9780470027318.a9258.pub2

Cited by 4 publications

(10 citation statements)

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“…1,2 Interaction between nucleosomes is a fundamental property that defines the assembly and function of chromatin. Studies over the past two decades have revealed highly dynamic features of nucleosomes that can explain regulatory processes at the chromatin level (e.g., see recent reviews [3][4][5] ). However, structural details and the mechanism underlying the assembly of nucleosomes in higher-order structures of chromatin and their dynamics remain unexplained.…”

Section: Introductionmentioning

confidence: 99%

Nanoscale Structure, Interactions, and Dynamics of Centromere Nucleosomes

Filliaux,

Sun,

Lyubchenko

2024

Preprint

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Centromeres are specific segments of chromosomes responsible for the accurate chromosome segregation process. Centromeres are comprised of two types of nucleosomes: canonical nucleosomes containing an octamer of H2A, H2B, H3, and H4 histones, and CENP-A nucleosomes in which H3 is replaced with its analog CENP-A histone. This modification leads to the difference in the nuclear of DNA turns around the histone core, wrapping efficiency. This value is 121 bp of DNA, considerably less than 147 bp found in canonical nucleosomes. We used Atomic Force Microscopy (AFM) to characterize nanoscale features for both types of nucleosomes assembled on the same template, enabling us to evaluate the effect of internucleosomal interaction. We found that CENP-A mononucleosomes have a lower internucleosomal affinity than canonical H3 nucleosomes. We applied time-lapse, high-speed AFM (HS-AFM) to characterize the dynamics of nucleosomes. For both nucleosomes, spontaneous unwrapping of DNA was observed, and this process occurs via a transient state with ~100 bp DNA wrapped around the core, followed by a rapid dissociation of DNA. The unwrapping process is asymmetric, so when the dissociation starts on one arm, it enlarges the size of the dissociated arm. Additionally, HS-AFM revealed higher stability of CENP-nucleosomes compared with H3 ones, in which dissociation of the histone core occurs prior to the nucleosome dissociation. The histone core of CENP-A nucleosomes remains intact even after the dissociation of DNA.

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Section: Introductionmentioning

confidence: 99%

Nanoscale Structure, Interactions, and Dynamics of Centromere Nucleosomes

Filliaux,

Sun,

Lyubchenko

2024

Preprint

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“…After measuring the thickness with the surface profilometer, the thermal evaporation tooling factor is adjusted to achieve optimum thickness. There are three operating modes for an AFM; contact mode, tapping mode, and non-contact modes [159]. In contact mode, there is physical contact between the probe and the sample surface.…”

Section: Surface Profilometermentioning

confidence: 99%

“…Despite contact mode offering high-speed scanning with high resolution, there is a higher chance of damaging the tip and the sample surface during scanning. Therefore, contact mode is usually used for flat and rigid surfaces [159]. In this thesis, surface roughness measurements are carried out in contact operating mode.…”

Section: (A) (B)mentioning

confidence: 99%

“…By keeping the change in amplitude position at a constant value with the help of a feedback loop, a topographical image is achieved without any damage to the tip on the sample surface. Due to the low applied force and high-resolution images, tapping mode is successfully used in the topographical characterisation of samples [159].…”

Section: (A) (B)mentioning

confidence: 99%

“…Monitoring the variation of the resonant frequency of the probe and free oscillation of the system gives atomic tipsample interaction force intensities and produces topographic images. This operating mode is mostly used in soft materials such as biological tissues [159].…”

Section: (A) (B)mentioning

confidence: 99%

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Bimetallic structures to enhance the performance of surface plasmon resonance sensors

Babu¹

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Surface plasmon resonance (SPR) sensing is a label−free and rapid detection method and has extensive applications in the field of medical diagnostics, food control, and environmental monitoring. However, the lack of sensitivity to detect small molecules is a continuing concern in the application of this technique. Past research has explored different plasmonic structures such as metal nanoparticles, metallic nanoslits, nanoholes, colloidal Au nanoparticles, 2D nanomaterials, and multilayer structures as the sensing layer to improve the sensitivity of these sensors. However, the sensitivity improvement could be realised only with the cost of the increased complexity of optical configuration and sensor chip fabrication. Silver (Ag) is a very good candidate as the metallic layer for the sensor chip due to its higher electrical conductivity as compared to gold (Au). Besides cost−effectiveness, Ag thin film based sensors have better sensitivity with a sharp resonance dip and a high signal−to−noise ratio. However, the poor chemical stability of Ag thin films prevents their use in practical applications. Noble metals such as Au and platinum (Pt) offer greatly enhanced chemical stability. This work investigated the development of SPR sensors composed of a silver−noble metal bilayer structure to utilize both the sensitivity of silver and the chemical stability of the noble metal. To enable this research, an automated experimental SPR testbed for sensor characterisation was designed and constructed. This testbed is based on the Kretschmann configuration, using a He−Ne laser source at 632.8 nm. SPR sensor consisting of multilayer metal structures was fabricated using standard microelectronic fabrication techniques. The influence of the relative thickness of a noble metal capping layer on the SPR response and sensitivity from the Ag layer was systematically optimised, using both theoretical and experimental approaches. A theoretical analysis of the performance of the bimetallic SPR sensors was done using the transfer matrix method (TMM) by assuming a five−layer configuration. In the case of an Au capping layer, these simulations indicate an optimised thickness of 45 nm for Ag and 5 nm for Au. The observation from experimental analysis of different thickness combinations of Ag and Au matched the simulated results. However, the results of the stability studies exclude the practical use of 45 nm Ag/5 nm Au structures, as long−term degradation of the Ag layer occurs. A structure of 40 nm Ag/10nm Au was thus selected as the best composition for sensor applications. It is showed that sensors fabricated with this structure showed enhanced sensitivity compared to single−layer Au sensors, with a sensitivity 50% higher than that of the single−layer Au sensor. In the case of Ag/Pt structures, simulations indicated enhanced sensitivity from a 10 nm Ag/16 nm Pt structure. However, experimental measurements did not show any evidence for SPP excitation of Pt at the measured wavelength of 632.8 nm, making it unsuitable as a capping layer in our studies. The application of 40 nm Ag/10 nm Au bimetal layers as biosensors was done by the immobilization of thiol−terminated vitamin B12 aptamers on the Au sensor surface. However, the results were not reproducible, and more work on the binding kinetics of this aptamer will need to be performed to use this in a biosensor structure.

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Nanoscale Structure, Interactions, and Dynamics of Centromere Nucleosomes

Filliaux,

Sun,

Lyubchenko

2024

Biomacromolecules

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Centromeres are specific segments of chromosomes comprising two types of nucleosomes: canonical nucleosomes containing an octamer of H2A, H2B, H3, and H4 histones and CENP-A nucleosomes in which H3 is replaced with its analogue CENP-A. This modification leads to a difference in DNA wrapping (∼121 bp), considerably less than 147 bp in canonical nucleosomes. We used atomic force microscopy (AFM) and high-speed AFM (HS-AFM) to characterize nanoscale features and dynamics for both types of nucleosomes. For both nucleosomes, spontaneous asymmetric unwrapping of DNA was observed, and this process occurs via a transient state with ∼100 bp DNA wrapped around the core, followed by a rapid dissociation of DNA. Additionally, HS-AFM revealed higher stability of CENP-A nucleosomes compared with H3 nucleosomes in which dissociation of the histone core occurs prior to the nucleosome dissociation. These results help elucidate the differences between these nucleosomes and the potential biological necessity for CENP-A nucleosomes.

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Atomic Force Microscopy Methods forDNAAnalysis

Cited by 4 publications

References 154 publications

Nanoscale Structure, Interactions, and Dynamics of Centromere Nucleosomes

Nanoscale Structure, Interactions, and Dynamics of Centromere Nucleosomes

Bimetallic structures to enhance the performance of surface plasmon resonance sensors

Nanoscale Structure, Interactions, and Dynamics of Centromere Nucleosomes

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