AFM-based single-molecule observation of the conformational changes of DNA structures

Endo, Masayuki

doi:10.1016/j.ymeth.2019.04.007

Cited by 14 publications

(9 citation statements)

References 53 publications

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“…Some of the earliest investigations using molecular mechanics employed the FLEX force field in combination with the energy optimization program JUMNA (junction minimization of NMR imino-H 1−5 ms for AT bp 38 -exact structure being monitored is not known exchange studies 10−50 ms for GC bp 38 -rate may be the rate of base wobbling 2,43−45,67 (monitor dynamics) 91−122 ms for AT tracts 68 -uncertain whether the target base, <5 ms for GC tracts 69 or its partner base, or both, have flipped out 2 FCS/ddFCS 52 even in presence of enzymes, -probe may not be specific to a base 2 (monitor dynamics) the lifetime obtained is of -alteration of natural structure of DNA by 70 insertion of probes the order of seconds. 46−50 -indirect observation since probe is placed 0.3−20 s for GT mismatched bp 53 on base adjacent to the target base 71 AFM lifetime of closed bp even in -results obtained depend on the interactions (monitor dynamics absence of stacking interactions between the target molecule and the at nm resolution) is of the order of seconds 54 surface it is attached to during AFM study 72 host−guest around 1000 s using -difficult to obtain base-specific host 77 This approach requires a predefined reaction coordinate (or order parameter), involving backbone torsions, distance and dihedral restraints. 43,67,78,79 For biomolecular reactions that involve a complex rearrangement of atoms, order parameters may introduce bias, 80 and regions of configuration space that are separated by large barriers can be incorrectly lumped together.…”

Section: ■ Introductionmentioning

confidence: 99%

Energy Landscapes for Base-Flipping in a Model DNA Duplex

2022

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We explore the process of base-flipping for four central bases, adenine, guanine, cytosine, and thymine, in a deoxyribonucleic acid (DNA) duplex using the energy landscape perspective. NMR imino-proton exchange and fluorescence correlation spectroscopy studies have been used in previous experiments to obtain lifetimes for bases in paired and extrahelical states. However, the difference of almost 4 orders of magnitude in the base-flipping rates obtained by the two methods implies that they are exploring different pathways and possibly different open states. Our results support the previous suggestion that minor groove opening may be favored by distortions in the DNA backbone and reveal links between sequence effects and the direction of opening, i.e., whether the base flips toward the major or the minor groove side. In particular, base flipping along the minor groove pathway was found to align toward the 5′ side of the backbone. We find that bases align toward the 3′ side of the backbone when flipping along the major groove pathway. However, in some cases for cytosine and thymine, the base flipping along the major groove pathway also aligns toward the 5′ side. The sequence effect may be caused by the polar interactions between the flipping-base and its neighboring bases on either of the strands. For guanine flipping toward the minor groove side, we find that the equilibrium constant for opening is large compared to flipping via the major groove. We find that the estimated rates of base opening, and hence the lifetimes of the closed state, obtained for thymine flipping through small and large angles along the major groove differ by 6 orders of magnitude, whereas for thymine flipping through small angles along the minor groove and large angles along the major groove, the rates differ by 3 orders of magnitude.

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Section: ■ Introductionmentioning

confidence: 99%

Energy Landscapes for Base-Flipping in a Model DNA Duplex

2022

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“…To manipulate single DNA molecules at the nanolevel, a DNA-origami structure, called a DNA-origami frame, was designed and built by the group of Paul Rothemund et al The DNA origami frame can accommodate two different DNA fragments in the cavity, resulting in incorporating any modified DNA and RNA strands [101]. The physical form such as tensions, rotations, and the torsion of incorporated DNA can be manipulated by inducing two different DNA strands into four connection sites in the DNA-origami frame [102]. In addition, the orientations of two dsDNA strands or four ssDNA strands can be manipulated via the DNA-origami frame, resulting in manipulating the arrangement of the incorporated DNA strands.…”

Section: Dna Origami and Control Of Dna Foldingmentioning

confidence: 99%

“…In addition, the orientations of two dsDNA strands or four ssDNA strands can be manipulated via the DNA-origami frame, resulting in manipulating the arrangement of the incorporated DNA strands. The four different ssDNA strands, three-way branched strand, and four-way branched strands formed in the DNA-origami frame have been used for the formation of the G-quadruplex [102][103][104], a substrate of proteins acting on DNA [88,89,105].…”

Section: Dna Origami and Control Of Dna Foldingmentioning

confidence: 99%

DNA Manipulation and Single-Molecule Imaging

2021

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DNA replication, repair, and recombination in the cell play a significant role in the regulation of the inheritance, maintenance, and transfer of genetic information. To elucidate the biomolecular mechanism in the cell, some molecular models of DNA replication, repair, and recombination have been proposed. These biological studies have been conducted using bulk assays, such as gel electrophoresis. Because in bulk assays, several millions of biomolecules are subjected to analysis, the results of the biological analysis only reveal the average behavior of a large number of biomolecules. Therefore, revealing the elementary biological processes of a protein acting on DNA (e.g., the binding of protein to DNA, DNA synthesis, the pause of DNA synthesis, and the release of protein from DNA) is difficult. Single-molecule imaging allows the analysis of the dynamic behaviors of individual biomolecules that are hidden during bulk experiments. Thus, the methods for single-molecule imaging have provided new insights into almost all of the aspects of the elementary processes of DNA replication, repair, and recombination. However, in an aqueous solution, DNA molecules are in a randomly coiled state. Thus, the manipulation of the physical form of the single DNA molecules is important. In this review, we provide an overview of the unique studies on DNA manipulation and single-molecule imaging to analyze the dynamic interaction between DNA and protein.

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“…Furthermore, by the chemical composition of the buffer interactions between the sample and substrate can be modified. Such surface modifications are often critical for successful AFM observations of protein structures and their functional motions ( Shlyakhtenko et al, 2010 ; Yamamoto et al, 2010 ; Endo, 2019 ).…”

Section: Introductionmentioning

confidence: 99%

Predicting the placement of biomolecular structures on AFM substrates based on electrostatic interactions

Amyot,

Nakamoto,

Kodera

et al. 2023

Front. Mol. Biosci.

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Atomic force microscopy (AFM) and high-speed AFM allow direct observation of biomolecular structures and their functional dynamics. Based on scanning the molecular surface of a sample deposited on a supporting substrate by a probing tip, topographic images of its dynamic shape are obtained. Critical to successful AFM observations is a balance between immobilization of the sample while avoiding too strong perturbations of its functional conformational dynamics. Since the sample placement on the supporting substrate cannot be directly controlled in experiments, the relative orientation is a priori unknown, and, due to limitations in the spatial resolution of images, difficult to infer from a posteriori analysis, thus hampering the interpretation of measurements. We present a method to predict the macromolecular placement of samples based on electrostatic interactions with the AFM substrate and demonstrate applications to HS-AFM observations of the Cas9 endonuclease, an aptamer-protein complex, the Monalysin protein, and the ClpB molecular chaperone. The model also allows predictions of imaging stability taking into account buffer conditions. We implemented the developed method within the freely available BioAFMviewer software package. Predictions based on available structural data can therefore be made even prior to an actual experiment, and the method can be applied for post-experimental analysis of AFM imaging data.

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AFM-based single-molecule observation of the conformational changes of DNA structures

Cited by 14 publications

References 53 publications

Energy Landscapes for Base-Flipping in a Model DNA Duplex

Energy Landscapes for Base-Flipping in a Model DNA Duplex

DNA Manipulation and Single-Molecule Imaging

Predicting the placement of biomolecular structures on AFM substrates based on electrostatic interactions

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