Dynamics and inhibition of MLL1 CXXC domain on DNA revealed by single-molecule quantification

Liang, Lin; Ma, Kangkang; Wang, Zeyu; Janissen, Richard; Yu, Zhongbo

doi:10.1016/j.bpj.2021.03.045

Cited by 5 publications

(4 citation statements)

References 53 publications

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“…We employed custom‐made magnetic tweezers, [ 9b,11a,28 ] consisting of a microscopy system, motor‐controlling magnets, and a flow cell connected to a pump. Single‐molecule experiments were conducted using a microsphere‐DNA‐coverslip setup within a microfluidic chamber.…”

Section: Methodsmentioning

confidence: 99%

“…[ 7a,10 ] These tags allow for the application of forces on both ends of the molecule via affinity interactions, allowing the investigation of single‐molecule dynamics of nucleic acids, proteins, and their target drugs. [ 9b,11 ] However, these single‐molecule mechanical strategies use only one manipulation geometry, which corresponds to the backbone of the biological macromolecule, such as the phosphate backbone of nucleic acids or the peptide bond backbone of proteins. Due to the limitation of manipulation geometry, single‐molecule techniques are particularly suitable for manipulating one molecule.…”

Section: Background and Originality Contentmentioning

confidence: 99%

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A DNA Force Circuit for Exploring Protein‐Protein Interactions at the Single‐Molecule Level^†

Ma,

Xiong,

Wang

et al. 2024

Chin. J. Chem.

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Comprehensive SummaryProtein‐protein interactions (PPIs) play a crucial role in drug discovery and disease treatment. However, the development of effective drugs targeting PPIs remains challenging due to limited methodologies for probing their spatiotemporal anisotropy. Here, we propose a single‐molecule approach using a unique force circuit to investigate PPI dynamics and anisotropy under mechanical forces. Unlike conventional techniques, this approach enables the manipulation and real‐time monitoring of individual proteins at specific amino acids with defined geometry, offering insights into molecular mechanisms at the single‐molecule level. The DNA force circuit was constructed using click chemistry conjugation methods and genetic code expansion techniques, facilitating orthogonal conjugation between proteins and nucleic acids. The SET domain of the MLL1 protein and the tail of histone H3 were used as a model system to demonstrate the application of the DNA force circuit. With the use of atomic force microscopy and magnetic tweezers, optimized assembly procedures were developed. The DNA force circuit provides an exceptional platform for studying the anisotropy of PPIs and holds promise for advancing drug discovery research targeted at PPIs.

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Section: Methodsmentioning

confidence: 99%

Section: Background and Originality Contentmentioning

confidence: 99%

A DNA Force Circuit for Exploring Protein‐Protein Interactions at the Single‐Molecule Level^†

Ma,

Xiong,

Wang

et al. 2024

Chin. J. Chem.

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“…[11] Recently, theoretical understanding on the dynamics of ligand binding or protein-DNA interactions has been quickly evolving at singlemolecule level, [12] helping us interpret many key factors in the protein-DNA dynamics, such as the functions of enzymes, the CpG patterns, and CpG flanking sequences, among others. [7,9,13] Here, we used single-molecule magnetic tweezers to quantitatively examine the dynamics of a CXXC domain on the promoter DNA of the Hoxa9 gene. The Hoxa9 gene has been serving studies of epigenetic enzyme interactions as a model DNA.…”

Section: Background and Originality Contentmentioning

confidence: 99%

Reading Time and DNA Sequence Preference of TET3 CXXC Domain Revealed by Single‐Molecule Profiling^†

Wang

Cao

et al. 2023

Chin. J. Chem.

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Comprehensive SummaryRecognition of CpG dinucleotide DNA in epigenetic information flow plays a pivotal role for cellular differentiation and development. The TET3 CXXC domain binds to CpG DNA, serving a basic epigenetic information reading mechanism. During the selective recognition of a CpG motif by a CXXC domain from crowded binding sites in a gene sequence, the protein‐DNA interactions are beyond CpG dinucleotide. However, the selective binding dynamics of CpG within a long DNA context by epigenetic enzymes have been rarely exploited, which is hard for ensemble methods to probe. Here, we used single‐molecule magnetic tweezers to quantitatively examine the dynamics of TET3's CXXC domain on a Hoxa9 promoter DNA. Our single‐molecule binding profile revealed that CXXC‐DNA interactions involve both CpG motifs and their flanking sequences. The residence time of TET3 CXXC differs by about 1000 times in five distinguished CpG clusters in the context of a CpG island. Moreover, we performed multi‐state hidden Markov modeling analysis on the zipping/unzipping dynamics of a CpG hairpin, discovering TET3 CXXC's preference on CpG motifs regarding the –2 to +2 flanking bases. Our results shed light on the selective binding dynamics of a CXXC on a gene sequence, facilitating studies on epigenetic information reading mechanisms.

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“…Single-molecule force spectroscopy (SMFS) [1] based on optical tweezers (OTs), atomic force microscopy (AFM) or magnetic tweezers (MTs), has become a powerful method for revealing enormous insight into the dynamics of biomolecules, such as DNA/RNA structural transitions, [2][3][4] protein folding/unfolding dynamics, [5][6][7][8] receptor-ligand binding/unbinding processes [9,10] and DNAprotein interactions. [11,12] Generally, SMFS requires a good number of data sets to produce adequate sampling distributions of events and states in order to precisely extract the kinetic and thermodynamics information. Progress has been made in the improvement of both the throughput of SMFS experiments and the lifetime of each molecule through the upgrade of instruments [13][14][15][16][17] and sample preparation method, [18][19][20] making it possible to obtain large data sets through parallel or repeated measurements.…”

Section: Introductionmentioning

confidence: 99%

Combination of density-clustering and supervised classification for event identification in single-molecule force spectroscopy data

et al. 2023

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Single-molecule force spectroscopy (SMFS) measurements of the dynamics of biomolecules typically require identifying massive events and states from large data sets, such as extracting rupture forces from force-extension curves (FECs) in pulling experiments and identifying states from extension-time trajectories (ETTs) in force-clamp experiments. The former is often accomplished manually and hence is time-consuming and laborious while the latter is always impeded by the presence of base-line drift. In this study, we attempt to accurately and automatically identify the events and states from SMFS experiments with a machine learning approach, ACCESS (approach combining clustering and classification for event identification of SMFS). As demonstrated by analysis of a series of data sets, ACCESS can extract the rupture forces from FECs containing multiple unfolding steps and classify the rupture forces into the corresponding conformational transitions. Moreover, ACCESS successfully identifies the unfolded and folded states even though the ETTs display severe non-monotonic base-line drift. Besides, ACCESS is straightforward in use as it requires only three easy-to-interpret parameters. As such, we anticipate that ACCESS will be a useful, easy-to-implement and high-performance tool for event and state identification across a range of single-molecule experiments.

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Dynamics and inhibition of MLL1 CXXC domain on DNA revealed by single-molecule quantification

Cited by 5 publications

References 53 publications

A DNA Force Circuit for Exploring Protein‐Protein Interactions at the Single‐Molecule Level^†

A DNA Force Circuit for Exploring Protein‐Protein Interactions at the Single‐Molecule Level^†

Reading Time and DNA Sequence Preference of TET3 CXXC Domain Revealed by Single‐Molecule Profiling^†

Combination of density-clustering and supervised classification for event identification in single-molecule force spectroscopy data

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Dynamics and inhibition of MLL1 CXXC domain on DNA revealed by single-molecule quantification

Cited by 5 publications

References 53 publications

A DNA Force Circuit for Exploring Protein‐Protein Interactions at the Single‐Molecule Level†

A DNA Force Circuit for Exploring Protein‐Protein Interactions at the Single‐Molecule Level†

Reading Time and DNA Sequence Preference of TET3 CXXC Domain Revealed by Single‐Molecule Profiling†

Combination of density-clustering and supervised classification for event identification in single-molecule force spectroscopy data

Contact Info

Product

Resources

About

A DNA Force Circuit for Exploring Protein‐Protein Interactions at the Single‐Molecule Level^†

A DNA Force Circuit for Exploring Protein‐Protein Interactions at the Single‐Molecule Level^†

Reading Time and DNA Sequence Preference of TET3 CXXC Domain Revealed by Single‐Molecule Profiling^†