-
Accounts of Chemical Research Aug 2022Facing increasing demand for precision medicine, materials chemistry systems for bioanalysis with accurate molecular design, controllable structure, and adjustable...
Facing increasing demand for precision medicine, materials chemistry systems for bioanalysis with accurate molecular design, controllable structure, and adjustable biological activity are required. As a genetic biomacromolecule, deoxyribonucleic acid (DNA) is created via precise, efficient, and mild processes in life systems and can in turn precisely regulate life activities. From the perspective of materials chemistry, DNA possesses the characteristics of sequence programmability and can be endowed with customized functions by the rational design of sequences. In recent years, DNA has been considered to be a potential biomaterial for analysis and has been applied in the fields of bioseparation, biosensing, and detection imaging. To further improve the precision of bioanalysis, the supramolecular assembly of DNA on micro/nanointerfaces is an effective strategy to concentrate functional DNA modules, and thus the functions of DNA molecules for bioanalysis can be enriched and enhanced. Moreover, the new modes of DNA supramolecular assembly on micro/nanointerfaces enable the integration of DNA with the introduced components, breaking the restriction of limited functions of DNA materials and achieving more precise regulation and manipulation in bioanalysis. In this Account, we summarize our recent work on DNA supramolecular assembly on micro/nanointerfaces for bioanalysis from two main aspects. In the first part, we describe DNA supramolecular assembly on the interfaces of microscale living cells. The synthesis strategy of DNA is based on rolling-circle amplification (RCA), which generates ultralong DNA strands according to circular DNA templates. The templates can be designed with complementary sequences of functional modules such as aptamers, which allow DNA to specifically bind with cellular interfaces and achieve efficient cell separation. In the second part, we describe DNA supramolecular assembly on the interfaces of nanoscale particles. DNA sequences are designed with functional modules such as targeting, drug loading, and gene expression and then are assembled on interfaces of particles including upconversion nanoparticles (UCNPs), gold nanoparticles (AuNPs), and magnetic nanoparticle (MNPs). The integration of DNA with these functional particles achieves cell manipulation, targeted tumor imaging, and cellular regulation. The processes of interfacial assembly are well controlled, and the functions of the obtained bioanalytical materials can be flexibly regulated. We envision that the work on DNA supramolecular assembly on micro/nanointerfaces will be a typical paradigm for the construction of more bioanalytical materials, which we hope will facilitate the development of precision medicine.
Topics: Biocompatible Materials; DNA; Gold; Metal Nanoparticles
PubMed: 35839123
DOI: 10.1021/acs.accounts.2c00170 -
Advances in Experimental Medicine and... 2020Clinical single-cell biomedicine has become a new emerging discipline, which integrates single-cell RNA and DNA sequencing, proteomics, and functions with clinical... (Review)
Review
Clinical single-cell biomedicine has become a new emerging discipline, which integrates single-cell RNA and DNA sequencing, proteomics, and functions with clinical phenomes, therapeutic responses, and prognosis. It is of great value to discover disease-, phenome-, and therapy-specific diagnostic biomarkers and therapeutic targets on the basis of the principle of clinical single-cell biomedicine. This book reviews the roles of single-cell sequencing and methylation in diseases and explores disease-specific alterations of single-cell sequencing and methylation, especially focusing on potential applications of methodologies on human single-cell sequencing and methylation, on potential correlations between those changes with pulmonary diseases, and on potential roles of signaling pathways that cause heterogeneous cellular responses during treatment. This book also emphasizes the importance of methodologies in clinical practice and application, the potential of perspectives, challenges and solutions, and the significance of single-cell preparation standardization. Alterations of DNA and RNA methylation, demethylation in lung diseases, and a deep knowledge about the regulation and function of target gene methylation for diagnosing and treating diseases at the early stage are also provided. Importantly, this book aims to apply the measurement of single-cell sequencing and methylation for clinical diagnosis and treatment and to understand clinical values of those parameters and to headline and foresee the potential values of the application of single-cell sequencing in non-cancer diseases.
Topics: DNA; DNA Methylation; Disease; Humans; Proteomics; RNA; Sequence Analysis; Single-Cell Analysis
PubMed: 32949386
DOI: 10.1007/978-981-15-4494-1_1 -
Journal of Translational Medicine Nov 2022Clinical CAR T-cell therapy using integrating vector systems represents a promising approach for the treatment of hematological malignancies. Lentiviral and...
BACKGROUND
Clinical CAR T-cell therapy using integrating vector systems represents a promising approach for the treatment of hematological malignancies. Lentiviral and γ-retroviral vectors are the most commonly used vectors in the manufacturing process. However, the integration pattern of these viral vectors and subsequent effect on CAR T-cell products is still unclear.
METHODS
We used a modified viral integration sites analysis (VISA) pipeline to evaluate viral integration events around the whole genome in pre-infusion CAR T-cell products. We compared the differences of integration pattern between lentiviral and γ-retroviral products. We also explored whether the integration sites correlated with clinical outcomes.
RESULTS
We found that γ-retroviral vectors were more likely to insert than lentiviral vectors into promoter, untranslated, and exon regions, while lentiviral vector integration sites were more likely to occur in intron and intergenic regions. Some integration events affected gene expression at the transcriptional and post-transcriptional level. Moreover, γ-retroviral vectors showed a stronger impact on the host transcriptome. Analysis of individuals with different clinical outcomes revealed genes with differential enrichment of integration events. These genes may affect biological functions by interrupting amino acid sequences and generating abnormal proteins, instead of by affecting mRNA expression. These results suggest that vector integration is associated with CAR T-cell efficacy and clinical responses.
CONCLUSION
We found differences in integration patterns, insertion hotspots and effects on gene expression vary between lentiviral and γ-retroviral vectors used in CAR T-cell products and established a foundation upon which we can conduct further analyses.
Topics: Humans; Lentivirus; Retroviridae; Genetic Vectors; Virus Integration; T-Lymphocytes; DNA
PubMed: 36348415
DOI: 10.1186/s12967-022-03729-5 -
ACS Nano Jul 2021DNA origami has emerged as a powerful molecular breadboard with nanometer resolution that can integrate the world of bottom-up (bio)chemistry with large-scale,... (Review)
Review
DNA origami has emerged as a powerful molecular breadboard with nanometer resolution that can integrate the world of bottom-up (bio)chemistry with large-scale, macroscopic devices created by top-down lithography. Substituting the top-down patterning with self-assembled colloidal nanoparticles now takes the manufacturing complexity of top-down lithography out of the equation. As a result, the deterministic positioning of single molecules or nanoscale objects on macroscopic arrays is benchtop ready and easily accessible.
Topics: DNA; Nanotechnology; Printing
PubMed: 34255962
DOI: 10.1021/acsnano.1c04297 -
G3 (Bethesda, Md.) Sep 2023In maize, the community-standard transformant line B104 is a useful model for dissecting features of transfer DNA (T-DNA) integration due to its compatibility with...
In maize, the community-standard transformant line B104 is a useful model for dissecting features of transfer DNA (T-DNA) integration due to its compatibility with Agrobacterium-mediated transformation and the availability of its genome sequence. Knowledge of transgene integration sites permits the analysis of the genomic environment that governs the strength of gene expression and phenotypic effects due to the disruption of an endogenous gene or regulatory element. In this study, we optimized a fusion primer and nested integrated PCR (FPNI-PCR) technique for T-DNA detection in maize to characterize the integration sites of 89 T-DNA insertions in 81 transformant lines. T-DNA insertions preferentially occurred in gene-rich regions and regions distant from centromeres. Integration junctions with and without microhomologous sequences as well as junctions with de novo sequences were detected. Sequence analysis of integration junctions indicated that T-DNA was incorporated via the error-prone repair pathways of nonhomologous (predominantly) and microhomology-mediated (minor) end-joining. This report provides a quantitative assessment of Agrobacterium-mediated T-DNA integration in maize with respect to insertion site features, the genomic distribution of T-DNA incorporation, and the mechanisms of integration. It also demonstrates the utility of the FPNI-PCR technique, which can be adapted to any species of interest.
Topics: Agrobacterium; Zea mays; Transformation, Genetic; DNA, Bacterial; DNA, Plant; Plants, Genetically Modified
PubMed: 37523773
DOI: 10.1093/g3journal/jkad166 -
Physical Review Letters Jul 2021DNA torsional elastic properties play a crucial role in DNA structure, topology, and the regulation of motor protein progression. However, direct measurements of these...
DNA torsional elastic properties play a crucial role in DNA structure, topology, and the regulation of motor protein progression. However, direct measurements of these parameters are experimentally challenging. Here, we present a constant-extension method integrated into an angular optical trap to directly measure torque during DNA supercoiling. We measured the twist persistence length of extended DNA to be 22 nm under an extremely low force (∼0.02 pN) and the twist persistence length of plectonemic DNA to be 24 nm. In addition, we implemented a rigorous data analysis scheme that bridged our measurements with existing theoretical models of DNA torsional behavior. This comprehensive set of torsional parameters demonstrates that at least 20% of DNA supercoiling is partitioned into twist for both extended DNA and plectonemic DNA. This work provides a new experimental methodology, as well as an analytical and interpretational framework, which will enable, expand, and enhance future studies of DNA torsional properties.
Topics: DNA; DNA, Superhelical; Elasticity; Models, Chemical; Nucleic Acid Conformation; Thermodynamics
PubMed: 34296898
DOI: 10.1103/PhysRevLett.127.028101 -
Accounts of Chemical Research Nov 2022G-quadruplexes (G4s) are distinctive four-stranded DNA or RNA structures found within cells that are thought to play functional roles in gene regulation and...
G-quadruplexes (G4s) are distinctive four-stranded DNA or RNA structures found within cells that are thought to play functional roles in gene regulation and transcription, translation, recombination, and DNA damage/repair. While G4 structures can be uni-, bi-, or tetramolecular with respect to strands, folded unimolecular conformations are most significant . Unimolecular G4 can potentially form in sequences with runs of guanines interspersed with what will become loops in the folded structure: 5'GLGLGLG, where is typically 2-4 and is highly variable. Such sequences are highly conserved and specifically located in genomes. In the folded structure, guanines from each run combine to form planar tetrads with four hydrogen-bonded guanine bases; these tetrads stack on one another to produce four strand segments aligned in specific parallel or antiparallel orientations, connected by the loop sequences. Three types of loops (lateral, diagonal, or "propeller") have been identified. The stacked tetrads form a central cavity that features strong coordination sites for monovalent cations that stabilize the G4 structure, with potassium or sodium preferred. A single monomeric G4 typically forms from a sequence containing roughly 20-30 nucleotides. Such short sequences have been the primary focus of X-ray crystallographic or NMR studies that have produced high-resolution structures of a variety of monomeric G4 conformations. These structures are often used as the basis for drug design efforts to modulate G4 function.We believe that the focus on monomeric G4 structures formed by such short sequences is perhaps myopic. Such short sequences for structural studies are often arbitrarily selected and removed from their native genomic sequence context, and then are often changed from their native sequences by base substitutions or deletions intended to optimize the formation of a homogeneous G4 conformation. We believe instead that G-quadruplexes prefer company and that in a longer natural sequence context multiple adjacent G4 units can form to combine into more complex multimeric G4 structures with richer topographies than simple monomeric forms. Bioinformatic searches of the human genome show that longer sequences with the potential for forming multiple G4 units are common. Telomeric DNA, for example, has a single-stranded overhang of hundreds of nucleotides with the requisite repetitive sequence with the potential for formation of multiple G4s. Numerous extended promoter sequences have similar potentials for multimeric G4 formation. X-ray crystallography and NMR methods are challenged by these longer sequences (>30 nt), so other tools are needed to explore the possible multimeric G4 landscape. We have implemented an integrated structural biology approach to address this challenge. This approach integrates experimental biophysical results with atomic-level molecular modeling and molecular dynamics simulations that provide quantitatively testable model structures. In every long sequence we have studied so far, we found that multimeric G4 structures readily form, with a surprising diversity of structures dependent on the exact native sequence used. In some cases, stable hairpin duplexes form along with G4 units to provide an even richer landscape. This Account provides an overview of our approach and recent progress and provides a new perspective on the G-quadruplex folding landscape.
Topics: Humans; G-Quadruplexes; DNA; Telomere; Guanine; Molecular Dynamics Simulation; Nucleotides; Nucleic Acid Conformation
PubMed: 36282946
DOI: 10.1021/acs.accounts.2c00519 -
Nano Letters Dec 2023Sophisticated dynamic molecular systems with diverse functions have been fabricated by using the fundamental tool of toehold-mediated strand displacement (TMSD) in the...
Sophisticated dynamic molecular systems with diverse functions have been fabricated by using the fundamental tool of toehold-mediated strand displacement (TMSD) in the field of dynamic DNA nanotechnology. However, simple approaches to reset these TMSD-based dynamic systems are lacking due to the difficulty in creating kinetically favored pathways to implement the backward resetting reactions. Here, we develop a facile proton-driven strategy to achieve complete resetting of a modular DNA circuit by integrating a pH-responsive intermolecular CG-C triplex DNA and an i-motif DNA into the conventional DNA substrate. The pH-programmed strategy allows modular DNA components to specifically associate/dissociate to promote the forward/backward TMSD reactions, thereby enabling the modular DNA circuit to be repeatedly operated at a constant temperature without generating any DNA waste products. Leveraging this tractable approach, we further constructed two resettable DNA logic gates used for logical computation and two resettable catalytic DNA systems with good performance in signal transduction and amplification.
Topics: DNA; DNA, Catalytic; Nanotechnology; Hydrogen-Ion Concentration
PubMed: 38085915
DOI: 10.1021/acs.nanolett.3c03265 -
Bioelectrochemistry (Amsterdam,... Oct 2022As an artificial nanomachine, a DNA walker demonstrates the potential for biosensing. In this study, a highly integrated, biostable, and autonomous electrochemical DNA...
As an artificial nanomachine, a DNA walker demonstrates the potential for biosensing. In this study, a highly integrated, biostable, and autonomous electrochemical DNA walker sensor was rationally designed by a simple assembly of a Mn-dependent DNAzyme-powered DNA walker with nanoscale Mn @MOFs containing free carboxylic acid groups UiO-66(Zr)-(COOH). In this study, the release of Mn from Mn@MOFs was exploited to drive the autonomous and progressive operation of the DNA walker, and the DNAzyme-driven DNA walker was constructed by the co-modification of walking strands and track strands onto the gold electrode (GE) surface. The walking strand was a single-stranded DNA containing a DNAzyme sequence, which was pre-silenced by the locking strand. The track strand was a specially designed DNA sequence that the target can hybridize with the locking strand; hence, the walking strand is unlocked, and the liberated DNAzyme catalyzes the cleavage of track strands to drive the DNA walker operation, shifting tetraferrocene away from the electrode and producing a significant signal change. A detection limit of 38 fM was obtained with our new system, exhibiting a wide linear range from 1.5625 × 10 M to 1 × 10 M. The proposed approach provided a novel means for constructing an highly integrated, automated, and DNAzyme-driven DNA walker for bioanalysis.
Topics: Biosensing Techniques; DNA; DNA, Catalytic; Electrochemical Techniques; Gold; Limit of Detection; Metal-Organic Frameworks; Phthalic Acids
PubMed: 35964550
DOI: 10.1016/j.bioelechem.2022.108198 -
Methods (San Diego, Calif.) Nov 2023The transcription, replication, packaging, and repair of genetic information ubiquitously involves DNA:protein interactions and other biological processes that require...
The transcription, replication, packaging, and repair of genetic information ubiquitously involves DNA:protein interactions and other biological processes that require local mechanical distortions of DNA. The energetics of such DNA-deforming processes are thus dependent on the local mechanical properties of DNA such as bendability or torsional rigidity. Such properties, in turn, depend on sequence, making it possible for sequence to regulate diverse biological processes by controlling the local mechanical properties of DNA. A deeper understanding of how such a "mechanical code" can encode broad regulatory information has historically been hampered by the absence of technology to measure in high throughput how local DNA mechanics varies with sequence along large regions of the genome. This was overcome in a recently developed technique called loop-seq. Here we describe a variant of the loop-seq protocol, that permits making rapid flexibility measurements in low-throughput, without the need for next-generation sequencing. We use our method to validate a previous prediction about how the binding site for the bacterial transcription factor Integration Host Factor (IHF) might serve as a rigid roadblock, preventing efficient enhancer-promoter contacts in IHF site containing promoters in E. coli, which can be relieved by IHF binding.
Topics: Bacterial Proteins; Escherichia coli; Base Sequence; Integration Host Factors; Promoter Regions, Genetic; DNA; DNA, Bacterial; Binding Sites
PubMed: 37769928
DOI: 10.1016/j.ymeth.2023.09.007