DNA Progression Research Articles

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

AbstractDespite 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.

Nanopore-based multiplexed, sensitive biomarker quantification using antibody-based rolling circle amplification

Biological nanopores are robust platforms for the identification of a range of biomolecules, for example, nucleic acid and peptides. Although progress in DNA and RNA sequencing has seen a lot of success in recent years, progress towards applying quantitative nanopore diagnostic tools has been impeded by a few challenges. One of the major bottlenecks is the inefficient capture of molecules into a pore at low concentrations, for example, fM-level biomarker detection for cancer diagnosis. Here we present an immuno-rolling circle amplification scheme for reliable multiplexed biomarker quantification from biological fluids using a biological nanopore sensor.

Self-Assembling Nucleic Acid Nanostructures Functionalized with Aptamers.

Researchers have worked for many decades to master the rules of biomolecular design that would allow artificial biopolymer complexes to self-assemble and function similarly to the diverse biochemical constructs displayed in natural biological systems. The rules of nucleic acid assembly (dominated by Watson-Crick base-pairing) have been less difficult to understand and manipulate than the more complicated rules of protein folding. Therefore, nucleic acid nanotechnology has advanced more quickly than de novo protein design, and recent years have seen amazing progress in DNA and RNA design. By combining structural motifs with aptamers that act as affinity handles and add powerful molecular recognition capabilities, nucleic acid-based self-assemblies represent a diverse toolbox for use by bioengineers to create molecules with potentially revolutionary biological activities. In this review, we focus on the development of self-assembling nucleic acid nanostructures that are functionalized with nucleic acid aptamers and their great potential in wide ranging application areas.

Nanopore-based sensing interface for single molecule electrochemistry

Nanopore techniques are experiencing a gallop since it walked out the notebook and show its charm on the science arena. The nanoscale pore offers a single-molecule resolution with a label-free and high-selective manner for the research of molecular structures, molecular dynamics, single-molecule reactions and for a variety of applications in biophysics and bionanotechnology. In this review, we introduce the construction of three types of nanopore platforms along with the latest progress in DNA sensing, structure and dynamics analysis of peptides/proteins, and the detection of redox reactions with new sensing mechanisms. Then, we depict nanopore data processing methods which provide an insight of data mining under the background of big data. We could fully expect the great impact of nanopore techniques on not only for DNA sequencing and sensing applications, but also in protein sequencing and clinical diagnostics.

Processive DNA synthesis is associated with localized decompaction of constitutive heterochromatin at the sites of DNA replication and repair

ABSTRACTConstitutive heterochromatin is considered as a functionally inert genome compartment, important for its architecture and stability. How such stable structure is maintained is not well understood. Here, we apply four different visualization schemes to label it and investigate its dynamics during DNA replication and repair. We show that replisomes assemble over the heterochromatin in a temporally ordered manner. Furthermore, heterochromatin undergoes transient decompaction locally at the active sites of DNA synthesis. Using selective laser microirradiation conditions that lead to damage repaired via processive DNA synthesis, we measured similarly local decompaction of heterochromatin. In both cases, we could not observe large-scale movement of heterochromatin to the domain surface. Instead, the processive DNA synthesis machinery assembled at the replication/repair sites. Altogether, our data are compatible with a progression of DNA replication/repair along the chromatin in a dynamic mode with localized and transient decompaction that does not globally remodels the whole heterochromatin compartment.

Development of 3D printed mesofluidic devices to elute and concentrate DNA.

To understand structural variation for personal genomics, an extensive ensemble of large DNA molecules will be required to span large structural variations. Nanocoding, a whole-genome analysis platform, can analyze large DNA molecules for the construction of physical restriction maps of entire genomes. However, handling of large DNA is difficult and a system is needed to concentrate large DNA molecules, while keeping the molecules intact. Insert technology was developed to protect large DNA molecules during routine cell lysis and molecular biology techniques. However, eluting and concentrating DNA molecules has been difficult in the past. Utilizing 3D printed mesofluidic device, a proof of principle system was developed to elute and concentrate lambda DNA molecules at the interface between a solution and a poly-acrylamide roadblock. The matrix allowed buffer solution to move through the pores in the matrix; however, it slowed down the progression of DNA in the matrix, since the molecules were so large and the pore size was small. Using fluorescence intensity of the insert, 84% of DNA was eluted from the insert and 45% of DNA was recovered in solution from the eluted DNA. DNA recovered was digested with a restriction enzyme to determine that the DNA molecules remained full length during the elution and concentration of DNA.

DNA damage in blood lymphocytes in patients after (177)Lu peptide receptor radionuclide therapy.

PurposeThe aim of the study was to investigate DNA double strand break (DSB) formation and its correlation with the absorbed dose to the blood lymphocytes of patients undergoing their first peptide receptor radionuclide therapy (PRRT) with 177Lu-labelled DOTATATE/DOTATOC.MethodsThe study group comprised 16 patients receiving their first PRRT. At least six peripheral blood samples were obtained before, and between 0.5 h and 48 h after radionuclide administration. From the time–activity curves of the blood and the whole body, residence times for blood self-irradiation and whole-body irradiation were determined. Peripheral blood lymphocytes were isolated, fixed with ethanol and subjected to immunofluorescence staining for colocalizing γ-H2AX/53BP1 DSB-marking foci. The average number of DSB foci per cell per patient sample was determined as a function of the absorbed dose to the blood and compared with an in vitro calibration curve established in our laboratory with 131I and 177Lu.ResultsThe average number of radiation-induced foci (RIF) per cell increased over the first 5 h after radionuclide administration and decreased thereafter. A linear fit from 0 to 5 h as a function of the absorbed dose to the blood agreed with our in vitro calibration curve. At later time-points the number of RIF decreased, indicating progression of DNA repair.ConclusionMeasurements of RIF and the absorbed dose to the blood after systemic administration of 177Lu may be used to obtain data on the individual dose–response relationships in vivo. Individual patient data were characterized by a linear dose-dependent increase and an exponential decay function describing repair.Electronic supplementary materialThe online version of this article (doi:10.1007/s00259-015-3083-9) contains supplementary material, which is available to authorized users.

Improvements in allelic discrimination of microsatellite markers using denaturing polyacrylamide gel electrophoresis.

Poor resolution, retarded progress of DNA through gels, and variable sizing of DNA fragments between and within gels hinder accurate genotyping of some simple sequence length polymorphism (SSLP) markers with the Perkin Elmer Applied Biosystems 377 Sequenator. These problems are similar to renaturation related problems observed in DNA sequencing gels. PCR products especially susceptible to these problems are shown to have higher melting temperatures (T(m)) than others. Gels containing increased concentrations of denaturants allow greater accuracy in allelic discrimination. This is especially beneficial where quantification is necessary.

The progress of DNA analyzing techniques and its impact on plant molecular systematics

Recent advances in manipulating nucleic acids have opened a new research field called plant molecular systematics. This short review provides an overview of molecular techniques which have been used in the analysis of DNA molecules for the study of plant systematics, with a special emphasis on PCR. The early application of DNA analysis, DNA/DNA hybridization, has not become popular with plant systematists, because of several disadvantages inherent in the method. The survey of restriction fragment length polymorphisms (RFLPs), on the contrary, has become one of the preferred methods used by plant molecular systematists, since the method is relatively easy to perform. Although unambiguous data can be obtained by both long-range restriction mapping and nucleotide sequencing, these approaches may have limited use in plant molecular systematics because of their laborious experimental procedures relying on conventional molecular cloning techniques. To date, PCR based analyses of the DNA molecule seem to be the most suitable experimental approach for plant molecular systematics. Several advantages of the method have changed both the quality and quantity of the DNA data. Further application of PCR to plant molecular systematics will open up a new era in the field.

Growth characteristics of batch-cultured activated sludge and its phosphate elimination capacity

Growth characteristics of the batch-cultured and continuously cultured activated sludge were compared. The batch-cultured activated sludge was found to show a far higher rate of glucose uptake but a comparable rate of true growth, that is, growth accompanied by synthesis of nucleic acids and proteins. A large portion of the glucose rapidly taken up is accumulated in sludge in the form of polyglucose and similar polysaccharides, which later decreases gradually with the progress of DNA, RNA and protein synthesis. A decline in DO, as a result of rapid oxygen absorption, was observed upon uptake of glucose. At the same time, certain metabolic functions were found to develop under anaerobic conditions, including an organics-uptake system accompanied by phosphate release. This development leads to the increase in the amount of polyphosphate in sludge, while the rapid uptake of organics not directly conjugate with growth correlates with bulking suppression.

Flow cytometry of mammalian sperm: progress in DNA and morphology measurement.

Variability in DNA content and head shape of mammalian sperm are potentially useful markers for flow cytometric monitoring of genetic damage in spermatogenic cells. The high refractive index and extreme flatness of the sperm heads produce an optical effect which interferes with DNA measurements in flow cytometers which have dye excitation and fluorescence light collection normal to the axis of flow. Orientation of sperm in flow controls this effect and results in coefficients of variation of 2.5% and 4.2%, respectively, for DNA measurements of mouse and human sperm. Alternatively, the optical effect can be used to generate shape-related information. Measurements on randomly oriented sperm from three mammalian species using a pair of fluorescence detectors indicate that large shape differences are detectable. Acriflavine-Feulgen stained sperm nuclei are significantly bleached during flow cytometric measurements at power levels routinely used in many flow cytometers. Dual beam studies of this phenomenon indicate it may be useful in detecting abnormally shaped sperm.