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Journal of the American Chemical Society Feb 2024Two-dimensional (2D) DNA origami assembly represents a powerful approach to the programmable design and construction of advanced 2D materials. Within the context of...
Two-dimensional (2D) DNA origami assembly represents a powerful approach to the programmable design and construction of advanced 2D materials. Within the context of hybridization-mediated 2D DNA origami assembly, DNA spacers play a pivotal role as essential connectors between sticky-end regions and DNA origami units. Here, we demonstrated that programming the spacer length, which determines the binding radius of DNA origami units, could effectively tune sticky-end hybridization reactions to produce distinct 2D DNA origami arrays. Using DNA-PAINT super-resolution imaging, we unveiled the significant impact of spacer length on the hybridization efficiency of sticky ends for assembling square DNA origami (SDO) units. We also found that the assembly efficiency and pattern diversity of 2D DNA origami assemblies were critically dependent on the spacer length. Remarkably, we realized a near-unity yield of ∼98% for the assembly of SDO trimers and tetramers via this spacer-programmed strategy. At last, we revealed that spacer lengths and thermodynamic fluctuations of SDO are positively correlated, using molecular dynamics simulations. Our study thus paves the way for the precision assembly of DNA nanostructures toward higher complexity.
Topics: DNA, Intergenic; Nucleic Acid Conformation; DNA; Nanostructures; Nucleic Acid Hybridization; Nanotechnology
PubMed: 38355136
DOI: 10.1021/jacs.3c13180 -
International Journal of Molecular... May 2024Nucleic acids are not only static carriers of genetic information but also play vital roles in controlling cellular lifecycles through their fascinating structural...
Nucleic acids are not only static carriers of genetic information but also play vital roles in controlling cellular lifecycles through their fascinating structural diversity [...].
Topics: RNA; DNA; Computational Biology; Nucleic Acid Conformation; Humans
PubMed: 38791265
DOI: 10.3390/ijms25105226 -
Methods (San Diego, Calif.) Nov 2023The development of compounds that can selectively bind with non-canonical DNA structures has expanded in recent years. Junction DNA, including three-way junctions (3WJs)...
The development of compounds that can selectively bind with non-canonical DNA structures has expanded in recent years. Junction DNA, including three-way junctions (3WJs) and four-way Holliday junctions (HJs), offer an intriguing target for developmental therapeutics as both 3WJs and HJs are involved in DNA replication and repair processes. However, there are a limited number of assays available for the analysis of junction DNA binding. Here, we describe the design and execution of multiplex fluorescent polyacrylamide gel electrophoresis (PAGE) and microscale thermophoresis (MST) assays that enable evaluation of junction-binding compounds. Two well characterised junction-binding compounds-a C6 linked bis-acridine ligand and an iron(II)-bound peptide helicate, which recognise HJs and 3WJs, respectively-were employed as probes for both MST and PAGE experiments. The multiplex PAGE assay expands beyond previously reported fluorescent PAGE as it uses four individual fluorophores that can be combined to visualise single-strands, pseudo-duplexes, and junction DNA present during 3WJ and HJ formation. The use of MST to identify the binding affinity of junction binding agents is, to our knowledge, first reported example of this technique. The combined use of PAGE and MST provides complementary results for the visualisation of 3WJ and HJ formation and the direct binding affinity (K and EC) of these agents. These assays can be used to aid the discovery and design of new therapeutics targeting non-canonical nucleic acid structures.
Topics: DNA; DNA, Cruciform; DNA Replication; Electrophoresis, Polyacrylamide Gel
PubMed: 37690737
DOI: 10.1016/j.ymeth.2023.09.002 -
Analytical Chemistry Aug 2023The antitumor effect of Pt-based drugs is determined by their binding activity with deoxyribonucleic acid (DNA), and understanding the reaction process in a systematic...
The antitumor effect of Pt-based drugs is determined by their binding activity with deoxyribonucleic acid (DNA), and understanding the reaction process in a systematic manner is crucial. However, existing assays used for DNA-Pt research suffer from several issues, such as complicated sample preparation, preamplification, and expensive instruments, which dramatically limit their practical application. In this study, a novel method was presented to investigate the adducts of DNA and oxaliplatin using an α-hemolysin nanopore sensor. This approach allows for real-time monitoring of the DNA-oxaliplatin condensation process through the detection of nanopore events associated with DNA-oxaliplatin adducts. Specifically, type I and II signals exhibiting specific current characteristics were observed during the process. Typical signals with high frequency were obtained by recording the designed DNA sequence. Furthermore, the production of these signals was confirmed to be independent of homologous adducts. This finding suggests that the DNA-oxaliplatin adduct can serve as a potential sensor for detecting oxaliplatin lesions and multiple types of molecules.
Topics: Oxaliplatin; Antineoplastic Agents; Hemolysin Proteins; Nanopores; DNA Adducts; Organoplatinum Compounds
PubMed: 37417945
DOI: 10.1021/acs.analchem.3c00461 -
F&S Science Aug 2023To determine the mechanistic role of mobile genetic elements in causing widespread DNA damage in primary human trophoblasts.
OBJECTIVE
To determine the mechanistic role of mobile genetic elements in causing widespread DNA damage in primary human trophoblasts.
DESIGN
Experimental ex vivo study.
SETTING
Hospital-affiliated University.
PATIENT(S)
Trophoblasts from a patient with unexplained recurrent pregnancy loss and patients with spontaneous and elective abortions (n = 10).
INTERVENTION(S)
Biochemical and genetic analysis and modification of primary human trophoblasts.
MAIN OUTCOME MEASURE(S)
To phenotype and systematically evaluate the underlying pathogenic mechanism for elevated DNA damage observed in trophoblasts derived from a patient with unexplained recurrent pregnancy loss, transcervical embryoscopy, G-band karyotyping, RNA sequencing, quantitative polymerase chain reaction, immunoblotting, biochemical and siRNA assays, and whole-genome sequencing were performed.
RESULT(S)
Transcervical embryoscopy revealed a severely dysmorphic embryo that was euploid on G-band karyotyping. RNA sequencing was notable for markedly elevated LINE-1 expression, confirmed with quantitative polymerase chain reaction, and that resulted in elevated expression of LINE-1-encoded proteins, as shown by immunoblotting. Immunofluorescence, biochemical and genetic approaches demonstrated that overexpression of LINE-1 caused reversible widespread genomic damage and apoptosis.
CONCLUSION(S)
Derepression of LINE-1 elements in early trophoblasts results in reversible but widespread DNA damage.
Topics: Pregnancy; Female; Humans; Trophoblasts; Retroelements; Abortion, Induced; Abortion, Habitual; Fetoscopy
PubMed: 37225003
DOI: 10.1016/j.xfss.2023.05.005 -
Transcription 2024RNA polymerases are the central enzymes of gene expression and function frequently in either a head-on or co-directional manner on the busy DNA track. Whether and how... (Review)
Review
RNA polymerases are the central enzymes of gene expression and function frequently in either a head-on or co-directional manner on the busy DNA track. Whether and how these collisions between RNA polymerases contribute to transcriptional regulation is mysterious. Increasing evidence from biochemical and single-molecule studies suggests that RNA polymerase collisions function as an important regulator to fine-tune transcription, rather than creating deleterious "traffic jams". This review summarizes the recent progress on elucidating the consequences of RNA polymerase collisions during transcription and highlights the significance of cooperation and coordination between RNA polymerases.
Topics: DNA; DNA-Directed RNA Polymerases; Transcription, Genetic
PubMed: 38357902
DOI: 10.1080/21541264.2024.2316972 -
The Journal of Physical Chemistry. B Jun 2024Tethered motion is ubiquitous in nature, offering controlled movement and spatial constraints to otherwise chaotic systems. The enhanced functionality and practical...
Tethered motion is ubiquitous in nature, offering controlled movement and spatial constraints to otherwise chaotic systems. The enhanced functionality and practical utility of tethers has been exploited in biotechnology, catalyzing the design of novel biosensors and molecular assembly techniques. While notable technological advances incorporating tethered motifs have been made, a theoretical gap persists within the paradigm, hindering a comprehensive understanding of tethered-based technologies. In this work, we focus on the characterization of the binding kinetics of two tethered molecules functionalized to a hard surface. Using a mean-field approximation, the binding time of such bimolecular system is determined analytically. Furthermore, estimates of the grafting site separation and polymer lengths which expedite binding are provided. These estimates, along with the analytical theories and frameworks established here, have the potential to improve efficacy in self-assembly methods in DNA nanotechnology and can be extended to more biologically specific endeavors including targeted drug-delivery and molecular sensing.
Topics: Kinetics; DNA; Nanotechnology; Polymers
PubMed: 38786364
DOI: 10.1021/acs.jpcb.4c01088 -
Cell Reports Nov 2023Abasic sites are common DNA lesions stalling polymerases and threatening genome stability. When located in single-stranded DNA (ssDNA), they are shielded from aberrant...
Abasic sites are common DNA lesions stalling polymerases and threatening genome stability. When located in single-stranded DNA (ssDNA), they are shielded from aberrant processing by 5-hydroxymethyl cytosine, embryonic stem cell (ESC)-specific (HMCES) via a DNA-protein crosslink (DPC) that prevents double-strand breaks. Nevertheless, HMCES-DPCs must be removed to complete DNA repair. Here, we find that DNA polymerase α inhibition generates ssDNA abasic sites and HMCES-DPCs. These DPCs are resolved with a half-life of approximately 1.5 h. HMCES can catalyze its own DPC self-reversal reaction, which is dependent on glutamate 127 and is favored when the ssDNA is converted to duplex DNA. When the self-reversal mechanism is inactivated in cells, HMCES-DPC removal is delayed, cell proliferation is slowed, and cells become hypersensitive to DNA damage agents that increase AP (apurinic/apyrimidinic) site formation. In these circumstances, proteolysis may become an important mechanism of HMCES-DPC resolution. Thus, HMCES-DPC formation followed by self-reversal is an important mechanism for ssDNA AP site management.
Topics: DNA Damage; Proteins; DNA Replication; DNA Repair; DNA; DNA, Single-Stranded
PubMed: 37950866
DOI: 10.1016/j.celrep.2023.113427 -
Methods in Molecular Biology (Clifton,... 2024DNA technique is a topic mandatorily covered in a biology and biochemistry undergraduate curriculum. Inquiry-based pedagogy is proven to be the most effective way of...
DNA technique is a topic mandatorily covered in a biology and biochemistry undergraduate curriculum. Inquiry-based pedagogy is proven to be the most effective way of learning, and DNA barcoding method allows to merge necessary-to-study experimental techniques such as DNA isolation and purification, PCR, and basic BLAST search into a two- or three-week inquiry-based student project. It also provides a research-based experience to the students, who, when organized in groups, can design their own DNA-barcoding project if they wish. Here, we describe how DNA barcoding can be offered in an undergraduate college or advanced high school settings. This chapter is intended to help college and high school instructors to include DNA barcoding in their classes.
Topics: DNA Barcoding, Taxonomic; Students; Universities; DNA; Humans; Curriculum; Polymerase Chain Reaction
PubMed: 38683341
DOI: 10.1007/978-1-0716-3581-0_33 -
Nature Chemical Biology Jun 2024
Topics: RNA; DNA Cleavage; DNA
PubMed: 38730193
DOI: 10.1038/s41589-024-01625-1