Flanking strand separation activity of RecA nucleoprotein filaments in DNA strand exchange reactions.

The recombinase RecA/Rad51 ATPase family proteins catalyze paramount DNA strand exchange reactions that are critically involved in maintaining genome integrity. However, it remains unclear how DNA strand exchange proceeds when encountering RecA-free defects in recombinase nucleoprotein filaments. Herein, by designing a series of unique substrates (e.g. truncated or conjugated incoming single-stranded DNA, and extended donor double-stranded DNA) and developing a two-color alternating excitation-modified single-molecule real-time fluorescence imaging assay, we resolve the two key steps (donor strand separation and new base-pair formation) that are usually inseparable during the reaction, revealing a novel long-range flanking strand separation activity of synaptic RecA nucleoprotein filaments. We further evaluate the kinetics and free energetics of strand exchange reactions mediated by various substrates, and elucidate the mechanism of flanking strand separation. Based on these findings, we propose a potential fundamental molecular model involved in flanking strand separation, which provides new insights into strand exchange mechanism and homologous recombination.

DNA Nanotechnology Targeting Mitochondria: From Subcellular Molecular Imaging to Tailor-Made Therapeutics.

Mitochondria, one of the most important organelles, represent a crucial subcellular target for fundamental research and biomedical applications. Despite significant advances in the design of DNA nanotechnologies for a variety of bio-applications, the dearth of strategies that enable mitochondria targeting for subcellular molecular imaging and therapy remains an outstanding challenge in this field. In this Minireview, we summarize the recent progresses on the emerging design and application of DNA nanotechnology for mitochondria-targeted molecular imaging and tumor treatment. We first highlight the engineering of mitochondria-localized DNA nanosensors for in situ detection and imaging of diverse key molecules that are essential to maintain mitochondrial functions, including mitochondrial DNA and microRNA, enzymes, small molecules, and metal ions. Then, we compile the developments of DNA nanotechnologies for mitochondria-targeted anti-tumor therapy, including modularly designed DNA nanodevices for subcellular delivery of therapeutic agents, and programmed DNA assembly for mitochondrial interference. We will place an emphasis on clarification of the chemical principles of how DNA nanobiotechnology can be designed to target mitochondria for various biomedical applications. Finally, the remaining challenges and future directions in this emerging field will be discussed, hoping to inspire further development of advanced DNA toolkits for both academic and clinical research regarding mitochondria.

Enzyme Translocation-Mediated Signal Amplification for Spatially Selective Aptasensing of ATP in Inflammatory Cells.

Amplified ATP imaging in inflammatory cells is highly desirable. However, the spatial selectivity of current amplification methods is limited, that is, signal amplification is performed systemically and not in a disease site-specific manner. Here we present a versatile strategy, termed enzymatically triggerable, aptamer-based signal amplification (ETA-SA), that enables inflammatory cell-specific imaging of ATP through spatially-resolved signal amplification. The ETA-SA leverages a translocated enzyme in inflammatory cells to activate DNA aptamer probes and further drive cascade reactions through the consumption of hairpin fuels, which, however, exerts no ATP response activity in normal cells, leading to a significantly improved sensitivity and spatial specificity for the inflammation-specific ATP imaging in vivo. Benefiting from the improved spatial selectivity, enhanced signal-to-background ratios were achieved for ATP imaging during acute hepatitis.

Photoactivated DNA Assembly and Disassembly for On-Demand Activation and Termination of cGAS-STING Signaling.

Despite significant progress in DNA self-assembly for interfacing with biology, spatiotemporally controlled regulation of biological process via in situ dynamic DNA assembly remains an outstanding challenge. Here, we report an optically triggered DNA assembly and disassembly strategy that enables on-demand activation and termination of the cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) signaling pathway. In the design, an activatable DNA hairpin is engineered with a photocleavable group at defined site to modulate its self-assembly activity. Light activation induces the configurational switching and consequent self-assembly of the DNA hairpins to form long linear double-stranded structures, allowing to stimulate cGAS protein to synthesize 2',3'-cyclic-GMP-AMP (cGAMP) for STING stimulation. Furthermore, by endowing the pre-assembled DNA scaffold with a built-in photolysis feature, we demonstrate that the cGAS-STING stimulation can be efficiently terminated through remote photo-triggering, providing for the first time a route to control the temporal "dose" on-demand for such a stimulation. We envision that this regulation strategy will benefit and inspire both fundamental research and therapeutic applications regarding the cGAS-STING pathway.

Spatially Selective Monitoring of Subcellular Enzyme Dynamics in Response to Mitochondria-Targeted Photodynamic Therapy.

Tracking spatial and temporal dynamics of bioactive molecules such as enzymes responding to therapeutic treatment is highly important for understanding of the related functions. However, in situ molecular imaging at subcellular level during photodynamic therapy (PDT) has been hampered by the limitations of existing methods. Herein, we present a multifunctional nanoplatform (termed as UR-HAPT) that is able to simultaneously monitor subcellular dynamics of human apurinic/apyrimidinic endonuclease 1 (APE1) during the near-infrared (NIR) light-mediated PDT. UR-HAPT was constructed by the combination of an upconversion nanoparticle-based PDT design and a mitochondria-targeting strategy with an APE1-responsive DNA reporter. Benefiting from the gain-of-function approach, activatable mitochondrial accumulation of APE1 in response to the oxidative stress was observed during the NIR light-triggered, mitochondria-targeted PDT process. We envision that this nanoplatform can be applicable to screen and evaluate potential enzyme inhibitors to improve the PDT efficacy.

Immunoassay of Small Molecule Mediated by a Triply Functional DNA.

Benefiting from specific target recognition by antibodies, the immunoassay is one of the widely used assays for the detection of biologically and environmentally important small molecules in broad fields. It can be challenge to isolate small molecules from their antibody complex in an immobilization-free immunoassay with separation for the detection of small-molecule targets. Here we present an immunoassay mediated by a triply functional DNA probe. A DNA strand is dually labeled with a fluorophore and the target small molecule. This DNA probe integrates three functions, including specific binding to the antibody, signal reporting for sensitive fluorescence detection, and carrying negative charges to facilitate capillary electrophoresis (CE) separation. The binding of the probe to an antibody brings many negative charges in the complex and causes significant changes in mass-to-charge ratios, so the antibody-probe complex can be well separated from the unbound probe in CE analysis. A simple immunoassay is achieved by target competition with this DNA probe for antibody binding in CE coupled to ultrasensitive laser-induced fluorescence (LIF) detection. To show a proof of concept, we detected two model small-molecule targets, digoxin, a therapeutic drug, and ochratoxin A (OTA), an important mycotoxin for food safety. In addition, the use of two DNA probes with distinguished migration times in CE allowed the simultaneous detection of OTA and digoxin. This immunoassay provides new opportunities for wide applications.

Profiling of the assembly of RecA nucleofilaments implies a potential target for environmental factors to disturb DNA repair.

Double-strand breaks (DSBs), one class of the most harmful DNA damage forms that bring elevated health risks, need to be repaired timely and effectively. However, an increasing number of environmental pollutants have been identified to impair DSB repair from various mechanisms. Our previous work indicated that the formation of unsaturated RecA nucleofilaments plays an essential role in homology recombination (HR) pathway which can accurately repair DSBs. In this study, by developing a benzonase cutting protection assay and combining it with traditional electrophoretic mobility shift assay (EMSA) analysis, we further investigated the assembly patterns of four RecA mutants that display differential DSB repair ability and ATPase activity. We observed that the mutants (G204S and S69G) possessing both ATP hydrolysis and DSB repair activities form unsaturated nucleofilaments similar to that formed by the wild type RecA, whereas the other two ATP hydrolysis-deficient mutants (K72R and E96D) that fail to mediate HR form more compacted nucleofilaments in the presence of ATP. These results establish a coupling of ATPase activity and effective DSB repair ability via the assembly status of RecA nucleofilaments. This linkage provides a potential target for environmental factors to disturb the essential HR pathway for DSB repair by suppressing the ATPase activity and altering the assembly pattern of nucleofilaments.

Quantitative assessments of adenosine triphosphatase hydrolytic activity by ultrafiltration-coupled ion-pair reversed-phase high-performance liquid chromatography.

Adenosine triphosphate is a universal energy currency that can directly provide energy required for a multitude of biochemical reactions and biophysical actions through adenosine triphosphatase catalyzed hydrolysis. Adenosine triphosphatase activity is thus one important feature for the characterization of protein function and cell activity. Herein, we optimized ion-pair reversed-phase high-performance liquid chromatography technique for highly efficient separation of adenosine triphosphate, adenosine diphosphate, and adenosine monophosphate, and the method demonstrated good linearity. Moreover, by coupling a protein-removable ultrafiltration, we developed a sensitive and robust approach for quantification of adenosine triphosphatase hydrolytic activity. By this assay, we demonstrated that RecA filaments-catalyzed adenosine triphosphate hydrolysis approached a second-order reaction, and its rate constant was estimated as 0.057 mM-1  min-1 . In addition, we explored the effects of DNA length on this reaction and revealed that the increase of the length of single-stranded DNA can promote the adenosine triphosphatase hydrolytic activity of RecA filaments. All these results confirm the feasibility of this new method in quantification of adenosine triphosphatase hydrolytic activity assays. Compared with previous complicated enzyme-coupled or homogeneous colorimetric measurements, the developed approach with high resolution separation allows a simple reaction system for adenosine triphosphatase assay and a sensitive detection free of interference from background noise.

GDx VCC measurements and blue-on-yellow perimetry in ocular hypertension.

The aim of this work was to investigate the correlation between scanning laser polarimetry with variable corneal compensation (GDx VCC) parameters and blue-on-yellow perimetry (B/YP) indices in ocular hypertension (OHT). One eye each of 52 patients with OHT (with a normal visual field, a normal optic nerve, and an untreated IOP > 21 mmHg) was chosen. All patients were examined with the Octopus 101 automated perimetry dG2 program using the dynamic/normal strategy (white-on-white perimetry, W/WP), with the dG2 program using the dynamic strategy/BY method (blue-on-yellow perimetry, B/YP), and with GDx VCC. Seven of the 52 OHT patients had both B/YP visual field loss and retinal nerve fiber layer (RNFL) defects with the GDx VCC measurements, and four patients demonstrated regions of B/YP visual field defects with corresponding RNFL defects on GDx VCC examination. A mild significant correlation was found between B/YP mean sensitivity (MS) and the superior average (R (2) =0.138, p = 0.049). No significant correlation was found between the W/WP indices and GDx VCC parameters. B/YP MS correlated with the superior average of GDx VCC in OHT patients. The findings brought up the possibility of using both techniques for clinical examination to yield converging data.

Precise sequencing of single protected-DNA fragment molecules for profiling of protein distribution and assembly on DNA.

Multiple DNA-interacting protein molecules are often dynamically distributed and/or assembled along a DNA molecule to adapt to their intricate functions temporally. However, analytical technology for measuring such binding behaviours is still missing. Here, we demonstrate the unique capacity of a supernuclease for a highly efficient cutting of the unprotected-DNA segments and with complete preservation of the protein-occluded DNA segments at near single-nucleotide resolution. By exploring this high-resolution cutting, an unprecedented assay that allows a precise sequencing of single protected-DNA fragment molecules (SPDFMS) was developed. As relevant applications, relevant information was gained on the respective distribution/assembly patterns and coordinated displacement of single-stranded DNA-binding protein and recombinase RecA, two model proteins, on DNA. Benefiting from this assay, we also for the first time provide direct measurement of the length of single RecA nucleofilaments, showing the predominant stoichiometry of 5-7 RecA monomers per RecA nucleofilament under physiologically relevant conditions. This innovative assay appears as a promising analytical tool for studying diverse protein-DNA interactions implicated in DNA replication, transcription, recombination, repair, and gene editing.

[Application of affinity capillary electrophoresis in the study of protein-DNA interactions].

Protein-DNA interactions play essential roles in various biological events that determine the cell fate. Research on the molecular mechanism of protein-DNA interactions has helped elucidate diverse fundamental life processes, thereby providing theoretical guidance for establishing clinical treatment and screening potential drug of target diseases. Furthermore, well-known protein-DNA interactions have been utilized to develop advanced bioengineering and bioanalytical techniques, therefore providing robust technical support for related research. Hence, it is important to establish sensitive and rapid analytical methods to study protein-DNA interactions. High-performance capillary electrophoresis (CE) has been widely used in many research fields such as chemistry, life sciences, and environmental sciences, mainly due to its advantages including ultra-high separation efficiency, extremely low sample consumption, and short analysis time. For instance, affinity capillary electrophoresis (ACE) has become an important analytical tool for investigating molecular interactions. In this paper, we review the applications of ACE in studying protein-DNA interactions since it was first proposed in 1992, addressing previous significant work in this field. Three major aspects have been summarized in this review: (1) brief introduction to the development of ACE technique; (2) applications of ACE in the fundamental research on the molecular mechanism of protein-DNA interactions; and (3) applications of well-known protein-DNA interactions in CE-based detection of target molecules and reactions. In the first aspect, along with the concept and separation modes of ACE, general strategies to enhance the analytical ability of ACE are briefly introduced. In the second aspect, the applications of ACE in studying several important protein-DNA interactions involving transcription factors (e.g., GCN4), DNA repair proteins (e.g., UvrA, UvrB, and RecA), and methylated DNA-binding proteins (MBDs) are reviewed. In the third aspect, the applications of well-known molecular interactions (e.g., antigen-antibody, aptamer-target, etc.) to facilitate CE-based detection of target molecules (e.g., DNA adducts, DNA methylation, microRNA, single nucleotide polymorphism, etc.) and target reactions (e.g., DNA strand exchange) are addressed. Finally, we prospect and discuss the advancements of ACE that can be established in future studies. The following two aspects should be improved in future ACE analysis: (1) the advantages of extremely low volume consumption and short analysis time should be fully utilized to develop sensitive and high-throughput CE platforms for the assessment of rare biological samples and massive uncertain samples, respectively; (2) ACE should be combined with other advanced techniques, such as DNA sequencing and mass spectrometry, to rapidly screen and identify the precise interacting sites of unknown protein-DNA interactions.

An electroosmotic flow-free two-direction migration strategy enables fast affinity capillary electrophoresis to study the weak interactions between basic peptides and RNA.

Affinity Capillary Electrophoresis (ACE) is a useful analytical tool to study noncovalent interactions. However, it remains challenging for ACE to measure weak and unstable interactions due to the fast dissociation of the binding complex and the possible destruction of the complex by a high electric field. In this study, we proposed a two-direction migration strategy that enables ACE to detect weak and unstable but important interactions by decreasing the migration distance of the binding complex and controlling the opposite migration direction of the free probe. By synthesizing a polyacrylamide-coated neutral capillary, free of electroosmotic flow, two-direction CE migration of basic peptides (positively charged) and peptide-RNA complexes (negatively charged) was achieved. Furthermore, the weak interactions between small nuclear U2 RNA and histone peptides were detected by this two-direction migration CE approach. The effects of the methylation states of histone peptides on the weak peptide-RNA interactions were also explored by this new approach. Collectively, the suggested modification of the ACE method is able to qualitatively characterize weak interactions.

High-affinity and undissociated capillary electrophoresis for DNA strand exchange analysis.

By identification of a super-stable protein-DNA-affinity system, we developed a free-solution capillary electrophoresis approach for rapid and sensitive detection of fundamentally important DNA strand exchange reactions mediated by recombinases. We further extended this assay for identification of hyper-recombinases generated from bioengineering and detection of single DNA mismatches caused by replication error.

ATPase activity tightly regulates RecA nucleofilaments to promote homologous recombination.

Homologous recombination (HR), catalyzed in an evolutionarily conserved manner by active RecA/Rad51 nucleofilaments, maintains genomic integrity and promotes biological evolution and diversity. The structures of RecA/Rad51 nucleofilaments provide information critical for the entire HR process. By exploiting a unique capillary electrophoresis-laser-induced fluorescence polarization assay, we have discovered an active form of RecA nucleofilament, stimulated by ATP hydrolysis, that contains mainly unbound nucleotide sites. This finding was confirmed by a nuclease protection assay and electron microscopy (EM) imaging. We further found that these RecA-unsaturated filaments promote strand exchange in vitro and HR in vivo. RecA mutants (P67D and P67E), which only form RecA-unsaturated nucleofilaments, were able to mediate HR in vitro and in vivo, but mutants favoring the formation of the saturated nucleofilaments failed to support HR. We thus present a new model for RecA-mediated HR in which RecA utilizes its intrinsic DNA binding-dependent ATPase activity to remodel the nucleofilaments to a less saturated form and thereby promote HR.

[Comparitive study of the threshold variability between blue-on-yellow perimetry and white-on-white perimetry].

PURPOSE: To compare the threshold variability of blue-on-yellow (B/Y) perimetry with white-white(W/W) perimetry, and evaluate the reproducibility of B/Y perimetry. METHODS: The B/Y perimetry and W/W perimetry in the Octopus 101 perimetry were used to examine the visual fields of 12 normal subjects (24 eyes), 16 cases (32 eyes) of primary open angle glaucoma (POAG), and 7 cases (14 eyes) of suspected POAG respectively. The B/Y perimetry and W/W perimetry were repeated to examine within 2 weeks. The point by point threshold variability of the two perimetries were compared and analysed. RESULTS: The total mean threshold variability for B/Y perimetry (2.61 +/- 0.94) dB was greater than that for W/W perimetry (2.11 +/- 0.90) dB in all subjects, but it had no significance (P = 0.6244). The total mean threshold variability for B/Y perimetry (2.07 +/- 0.54) dB was significantly greater than that for W/W perimetry (1.50 +/- 0.34) dB in normal subjects (P = 0.0006), while no significance was found in suspected POAG and POAG groups between the two perimetries (P = 0.0523 and 0.9371). The threshold variability in the areas of some eccentricities for B/Y perimetry were significantly greater than that for W/W perimetry in normal subjects and suspected POAG group, but no significance was found in POAG group for all eccentricities between the two perimetries. CONCLUSION: The threshold variability for B/Y perimetry was greater than that for W/W perimetry in normal subjects, but no significance was found in suspected POAG and POAG between the two perimetries.