Characterizing DNA Condensation and Conformational Changes in Organic Solvents

Organic solvents offer a new approach to formulate DNA into novel structures suitable for gene delivery. In this study, we examined the in situ behavior of DNA in N, N-dimethylformamide (DMF) at low concentration via laser light scattering (LLS), TEM, UV absorbance and Zeta potential analysis. Results revealed that, in DMF, a 21bp oligonucleotide remained intact, while calf thymus DNA and supercoiled plasmid DNA were condensed and denatured. During condensation and denaturation, the size was decreased by a factor of 8–10, with calf thymus DNA forming spherical globules while plasmid DNA exhibited a toroid-like conformation. In the condensed state, DNA molecules were still able to release the counterions to be negatively charged, indicating that the condensation was mainly driven by the excluded volume interactions. The condensation induced by DMF was reversible for plasmid DNA but not for calf thymus DNA. When plasmid DNA was removed from DMF and resuspended in an aqueous solution, the DNA was quickly regained a double stranded configuration. These findings provide further insight into the behavior and condensation mechanism of DNA in an organic solvent and may aid in developing more efficient non-viral gene delivery systems

Ion Competition in Condensed DNA Arrays in the Attractive Regime

AbstractPhysical origin of DNA condensation by multivalent cations remains unsettled. Here, we report quantitative studies of how one DNA-condensing ion (Cobalt3+ Hexammine, or Co3+Hex) and one nonDNA-condensing ion (Mg2+) compete within the interstitial space in spontaneously condensed DNA arrays. As the ion concentrations in the bath solution are systematically varied, the ion contents and DNA-DNA spacings of the DNA arrays are determined by atomic emission spectroscopy and x-ray diffraction, respectively. To gain quantitative insights, we first compare the experimentally determined ion contents with predictions from exact numerical calculations based on nonlinear Poisson-Boltzmann equations. Such calculations are shown to significantly underestimate the number of Co3+Hex ions, consistent with the deficiencies of nonlinear Poisson-Boltzmann approaches in describing multivalent cations. Upon increasing the concentration of Mg2+, the Co3+Hex-condensed DNA array expands and eventually redissolves as a result of ion competition weakening DNA-DNA attraction. Although the DNA-DNA spacing depends on both Mg2+ and Co3+Hex concentrations in the bath solution, it is observed that the spacing is largely determined by a single parameter of the DNA array, the fraction of DNA charges neutralized by Co3+Hex. It is also observed that only ∼20% DNA charge neutralization by Co3+Hex is necessary for spontaneous DNA condensation. We then show that the bath ion conditions can be reduced to one variable with a simplistic ion binding model, which is able to describe the variations of both ion contents and DNA-DNA spacings reasonably well. Finally, we discuss the implications on the nature of interstitial ions and cation-mediated DNA-DNA interactions

Postoperative serum squamous cell carcinoma antigen and carcinoembryonic antigen predict overall survival in surgical patients with esophageal squamous cell carcinoma

BackgroundTumor markers are routinely used in clinical practice. However, for resectable patients with esophageal squamous cell carcinoma (ESCC), they are applied infrequently as their prognostic significance is incompletely understood.MethodsThis historical cohort study included 2769 patients with resected ESCC from 2011 to 2018 in a high-risk area in northern China. Their clinical data were extracted from the Electronic Medical Record. Survival analysis of eight common tumor markers was performed with multivariable Cox proportional hazards regressions.ResultsWith a median follow-up of 39.5 months, 901 deaths occurred. Among the eight target markers, elevated postoperative serum SCC (Squamous cell carcinoma antigen) and CEA (Carcinoembryonic antigen) predicted poor overall survival (SCC HRadjusted: 2.67, 95% CI: 1.70-4.17; CEA HRadjusted: 2.36, 95% CI: 1.14-4.86). In contrast, preoperative levels were not significantly associated with survival. Stratified analysis also demonstrated poorer survival in seropositive groups of postoperative SCC and CEA within each TNM stage. The above associations were generally robust using different quantiles of concentrations above the upper limit of the clinical normal range as alternative cutoffs. Regarding temporal trends of serum levels, SCC and CEA were similar. Their concentrations fell rapidly after surgery and thereafter remained relatively stable.ConclusionPostoperative serum SCC and CEA levels predict the overall survival of ESCC surgical patients. More importance should be attached to the use of these markers in clinical applications

Nanoscale Structure and Interaction of Condensed Phases of DNA–Carbon Nanotube Hybrids

Condensation of DNA–carbon nanotube (CNT) hybrids dispersed in aqueous solutions can be induced by elevated hybrid concentrations, salts, or crowding agents. DNA–CNT condensates exhibit either nematic ordering or amorphous aggregates, dependent on the nature of interhybrid interactions. This study employed X-ray diffraction (XRD) to determine nanoscale structures of the condensates, including the presence of positional ordering, interaxial distances, and the range of ordered domains. To probe the effects of DNA sequence, two types of CNT hybrids, dispersed by genomic DNA of random sequence and synthetic oligonucleotides respectively, were studied under identical conditions. The osmotic stress method was further used to quantify force–distance dependencies of the DNA–CNT hybrids to elucidate the relation between interhybrid interactions and condensate structures. We observed that, independent of DNA sequence, lyotropic DNA–CNT phases showed weak positional ordering with long interhybrid distances, salt-induced condensates were amorphous, crowding-condensed DNA–CNTs were the most ordered with pronounced XRD peaks, and interhybrid interactions were defined by short-range hydration repulsion and long-range electrostatic repulsion. Conversely, the effects of DNA sequence became evident as to their quantitative force–distance relationships. Genomic DNA of random sequence consistently gave longer interhybrid distances than synthetic oligonucleotides, which we attribute to the likely differences in their hybrid diameters

Thermal denature curve of 21bp DNA fragment in 95% DMF.

<p>C = 20 µg/mL.</p

Thermal denature curves of calf thymus and plasmid DNA.

<p>Plasmid DNA: hollow symbols; calf thymus DNA: solid symbols. C = 16 µg/mL.</p

DMF-content dependence of zeta potential measurements of calf thymus DNA and plasmid DNA.

<p>DMF-content dependence of zeta potential measurements of calf thymus DNA and plasmid DNA.</p

Temperature effect on hydrodynamic radius of plasmid DNA in 95% DMF at 30°.

<p>The inset shows the temperature dependence of R_h,app at zero angle.</p

LLS studies on plasmid DNA (A: size distribution at 30°, B: angular dependence of excess scattered intensity) and calf thymus DNA (A': size distribution at 30°, B': angular dependence of excess scattered intensity).

<p>C = 1.6×10⁻⁵ g/ml.</p