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The cellular transport process of DNA is hampered by cell membrane barriers, and hence, a delivery vehicle is essential for realizing the potential benefits of gene therapy to combat a variety of genetic diseases. Virus-based vehicles are effective, although immunogenicity, toxicity and cancer formation are among the major limitations of this approach. Cationic polymers, such as polyethyleneimine are capable of condensing DNA to nanoparticles and facilitate gene delivery. Lack of biodegradation of polymeric gene delivery vehicles poses significant toxicity because of the accumulation of polymers in the tissue. Many attempts have been made to develop biodegradable polymers for gene delivery by modifying existing polymers and/or using natural biodegradable polymers. This review summarizes mechanistic aspects of gene delivery and the development of biodegradable polymers for gene delivery.

Polyamines are positively charged organic cations under physiologic ionic and pH conditions and hence they interact with negatively charged macromolecules such as DNA and RNA. Although electrostatic interaction is the predominant mode of polyamine–nucleic acid interactions, site- and structure-specific binding has also been recognized. A major consequence of polyamine–DNA interaction is the collapse of DNA to nanoparticles of approximately 100 nm diameter. Electron and atomic force microscopic studies have shown that these nanoparticles are spheroids, toroids and rods. DNA transport to cells for gene therapy applications requires the condensation of DNA to nanoparticles and hence the study of polyamines and related compounds with nucleic acids has received technological importance. In addition to natural and synthetic polyamines, several amine-terminated or polyamine-substituted agents are under intense investigation for non-viral gene delivery vehicles.

The imaging of individually labelled sister chromatids allows Nagasaka  et al.  to conclude that mitotic sister chromatin resolution begins in prophase and depends on the activity of topoisomerase II and condensin II, but not on cohesin dissociation. The formation of mitotic chromosomes requires both compaction of chromatin and the resolution of replicated sister chromatids. Compaction occurs during mitotic prophase and prometaphase, and in prophase relies on the activity of condensin II complexes 1 , 2 . Exactly when and how sister chromatid resolution occurs has been largely unknown, as has its molecular requirements. Here, we established a method to visualize sister resolution by sequential replication labelling with two distinct nucleotide derivatives. Quantitative three-dimensional imaging then allowed us to measure the resolution of sister chromatids throughout mitosis by calculating their non-overlapping volume within the whole chromosome. Unexpectedly, we found that sister chromatid resolution starts already at the beginning of prophase, proceeds concomitantly with chromatin compaction and is largely completed by the end of prophase. Sister chromatid resolution was abolished by inhibition of topoisomerase IIα and by depleting or preventing mitotic activation of condensin II, whereas blocking cohesin dissociation from chromosomes had little effect. Mitotic sister chromatid resolution is thus an intrinsic part of mitotic chromosome formation in prophase that relies largely on DNA decatenation and shares the molecular requirement for condensin II with prophase compaction.

Many viruses use molecular motors that generate large forces to package DNA to near-crystalline densities inside preformed viral proheads. Besides being a key step in viral assembly, this process is of interest as a model for understanding the physics of charged polymers under tight 3D confinement. A large number of theoretical studies have modeled DNA packaging, and the nature of the molecular dynamics and the forces resisting the tight confinement is a subject of wide debate. Here, we directly measure the packaging of single DNA molecules in bacteriophage phi29 with optical tweezers. Using a new technique in which we stall the motor and restart it after increasing waiting periods, we show that the DNA undergoes nonequilibrium conformational dynamics during packaging. We show that the relaxation time of the confined DNA is >10 min, which is longer than the time to package the viral genome and 60,000 times longer than that of the unconfined DNA in solution. Thus, the confined DNA molecule becomes kinetically constrained on the timescale of packaging, exhibiting glassy dynamics, which slows the motor, causes significant heterogeneity in packaging rates of individual viruses, and explains the frequent pausing observed in DNA translocation. These results support several recent hypotheses proposed based on polymer dynamics simulations and show that packaging cannot be fully understood by quasistatic thermodynamic models.

Background/Aim: Ally lisothiocyanate (AITC), a constituent of naturally occurring isothiocyanates (ITCs) found in some Brassica vegetables, has been previously demonstrated to have anti-carcinogenic activity. However, there is no available information showing that AITC induces DNA damage and alters DNA damage repair proteins in human breast cancer MCF-7 cells. Materials and Methods: In the present study, we investigated the effects of AITC on DNA damage and repair responses in human breast cancer MCF-7 cells in vitro. Cell viability was measured by flow cytometric assay. DNA condensation (apoptotic cell death) and DNA fragmentation (laddered DNA) were assayed by DAPI staining and DNA gel electrophoresis assays, respectively. Furthermore, DNA damage (comet tail) was measured by the comet assay. Western blotting was used to measure the expression of DNA damage- and repair-associated proteins. Results: AITC decreased cell viability in a dose-dependent and induced apoptotic cell death (DNA condensation and fragmentation) and DNA damage in MCF-7 cells. AITC increased p-ATMSer1981, p-ATRSer428, p53, p-p53Ser15, p-H2A.XSer139, BRCA1, and PARP at 10-30 μM at 24 and 48 h treatments. However, AITC decreased DNA-PK at 24 and 48 h treatment, and decreased MGMT at 48 h in MCF-7 cells. Conclusion: AITC induced cytotoxic effects (decreased viable cell number) through induction of DNA damage and condensation and altered DNA damage and repair associated proteins in MCF-7 cells in vitro.

Maslinic acid, a natural plant-derived triterpenoid compound, exhibits pharmacological activities, including anti-cancer. In the present study, we investigated the cytotoxic effects of maslinic acid on human cervical cancer HeLa cells in vitro and further investigated the molecular mechanism of maslinic acid-induced DNA damage and repair. Cell viability was measured by flow cytometry. DNA condensation (apoptotic cell death), DNA damage, and DNA fragmentation (DNA ladder) were assayed by DAPI staining, comet assay, and agarose gel electrophoresis, respectively. The expression of DNA damage and repair proteins was assayed by western blotting. Maslinic acid decreased total cell viability and induced DNA condensation, damage, and fragmentation in HeLa cells. Furthermore, maslinic acid elevated the levels of p-ATM , p-ATR , p53, p-p53 , p-H2A.X , BRCA1 and PARP at 30-40 μM. However, it decreased the levels of DNA-PK and MGMT. Maslinic acid reduced the number of viable HeLa cells by inducing DNA damage and altering the expression of proteins involved in DNA damage and repair.

In thermophoresis, with the fluid at rest, suspensions move along a gradient of temperature. In an aqueous solution, a PEG polymer suspension is depleted from the hot region and builds a concentration gradient. In this gradient, DNA polymers of different sizes can be separated. In this work the effect of the polymer structure for genomic DNA and small RNA is studied. For genome-size DNA, individual single T4 DNA is visualized and tracked in a PEG solution under a temperature gradient built by infrared laser focusing. We find that T4 DNA follows steps of depletion, ring-like localization, and accumulation patterns as the PEG volume fraction is increased. Furthermore, a coil–globule transition for DNA is observed for a large enough PEG volume fraction. This drastically affects the localization position of T4 DNA. In a similar experiment, with small RNA such as ribozymes we find that the stem–loop folding of such polymers has important consequences. The RNA polymers having a long and rigid stem accumulate, whereas a polymer with stem length less than 4 base pairs shows depletion. Such measurements emphasize the crucial contribution of the double-stranded parts of RNA for thermal separation and selection under a temperature gradient. Because huge temperature gradients are present around hydrothermal vents in the deep ocean seafloor, this process might be relevant, at the origin of life, in an RNA world hypothesis. Ribozymes could be selected from a pool of random sequences depending on the length of their stems.

Encapsulins are protein nanocages capable of harboring smaller proteins (cargo proteins) within their cavity. The function of the encapsulin systems is related to the encapsulated cargo proteins. The Myxococcus xanthus encapsulin (EncA) naturally encapsulates ferritin-like proteins EncB and EncC as cargo, resulting in a large iron storage nanocompartment, able to accommodate up to 30,000 iron atoms per shell. In the present manuscript we describe the binding and protection of circular double stranded DNA (pUC19) by EncA using electrophoretic mobility shift assays (EMSA), atomic force microscopy (AFM), and DNase protection assays. EncA binds pUC19 with an apparent dissociation constant of 0.3 ± 0.1 µM and a Hill coefficient of 1.4 ± 0.1, while EncC alone showed no interaction with DNA. Accordingly, the EncAC complex displayed a similar DNA binding capacity as the EncA protein. The data suggest that initially, EncA converts the plasmid DNA from a supercoiled to a more relaxed form with a beads-on-a-string morphology. At higher concentrations, EncA self-aggregates, condensing the DNA. This process physically protects DNA from enzymatic digestion by DNase I. The secondary structure and thermal stability of EncA and the EncA−pUC19 complex were evaluated using synchrotron radiation circular dichroism (SRCD) spectroscopy. The overall secondary structure of EncA is maintained upon interaction with pUC19 while the melting temperature of the protein (Tm) slightly increased from 76 ± 1 °C to 79 ± 1 °C. Our work reports, for the first time, the in vitro capacity of an encapsulin shell to interact and protect plasmid DNA similarly to other protein nanocages that may be relevant in vivo.

In all organisms, DNA molecules are tightly compacted into a dynamic 3D nucleoprotein complex. In bacteria, this compaction is governed by the family of nucleoid-associated proteins (NAPs). Under conditions of stress and starvation, an NAP called Dps (DNA-binding protein from starved cells) becomes highly up-regulated and can massively reorganize the bacterial chromosome. Although static structures of Dps–DNA complexes have been documented, little is known about the dynamics of their assembly. Here, we use fluorescence microscopy and magnetic-tweezers measurements to resolve the process of DNA compaction by Dps. Real-time in vitro studies demonstrated a highly cooperative process of Dps binding characterized by an abrupt collapse of the DNA extension, even under applied tension. Surprisingly, we also discovered a reproducible hysteresis in the process of compaction and decompaction of the Dps–DNA complex. This hysteresis is extremely stable over hour-long timescales despite the rapid binding and dissociation rates of Dps. A modified Ising model is successfully applied to fit these kinetic features. We find that long-lived hysteresis arises naturally as a consequence of protein cooperativity in large complexes and provides a useful mechanism for cells to adopt unique epigenetic states.