Kurt Andresen

Hinge stiffness is a barrier to RNA folding

Cation-mediated RNA folding from extended to compact, biologically active conformations relies on a temporal balance of forces. The Mg2 +-mediated folding of the Tetrahymena thermophila ribozyme is characterized by rapid nonspecific collapse followed by tertiary-contact-induced compaction. This article focuses on an autonomously folding portion of the Tetrahymena ribozyme, its P4-P6 domain, in order to probe one facet of the rapid collapse: chain flexibility. The time evolution of P4-P6 folding was followed by global and local measures as a function of Mg2 + concentration. While all concentrations of Mg2 + studied are sufficient to screen the charge on the helices, the rates of compaction and tertiary contact formation diverge as the concentration of Mg2 + increases; collapse is greatly accelerated by Mg2 +, while tertiary contact formation is not. These studies highlight the importance of chain stiffness to RNA folding; at 10 mM Mg2 +, a stiff hinge limits the rate of P4-P6 folding. At higher magnesium concentrations, the rate-limiting step shifts from hinge bending to tertiary contact formati

Mono- and trivalent ions around DNA: a small-angle scattering study of competition and interactions.

The presence of small numbers of multivalent ions in DNA-containing solutions results in strong attractive forces between DNA strands. Despite the biological importance of this interaction, e.g., DNA condensation, its physical origin remains elusive. We carried out a series of experiments to probe interactions between short DNA strands as small numbers of trivalent ions are included in a solution containing DNA and monovalent ions. Using resonant (anomalous) and nonresonant small angle x-ray scattering, we coordinated measurements of the number and distribution of each ion species around the DNA with the onset of attractive forces between DNA strands. DNA-DNA interactions occur as the number of trivalent ions increases. Surprisingly good agreement is found between data and size-corrected numerical Poisson-Boltzmann predictions of ion competition for non- and weakly interacting DNAs. We also obtained an estimate for the minimum number of trivalent ions needed to initiate DNA-DNA attraction.

Reconstructing three-dimensional shape envelopes from time-resolved small-angle X-ray scattering data

Modern computing power has made it possible to reconstruct low-resolution, three-dimensional shapes from solution small-angle X-ray scattering (SAXS) data on biomolecules without a priori knowledge of the structure. In conjunction with rapid mixing techniques, SAXS has been applied to time resolve conformational changes accompanying important biological processes, such as biomolecular folding. In response to the widespread interest in SAXS reconstructions, their value in conjunction with such time-resolved data has been examined. The group I intron from Tetrahymena thermophila and its P4-P6 subdomain are ideal model systems for investigation owing to extensive previous studies, including crystal structures. The goal of this paper is to assay the quality of reconstructions from time-resolved data given the sacrifice in signal-to-noise required to obtain sharp time resolution.

Focusing capillary optics for use in solution small-angle X-ray scattering

Measurements of the global conformation of macromolecules can be carried out using small-angle X-ray scattering (SAXS). Glass focusing capillaries, manufactured at the Cornell High Energy Synchrotron Source (CHESS), have been successfully employed for SAXS measurements on the heme protein cytochrome c. These capillaries provide high X-ray flux into a spot size of tens of micrometres, permitting short exposures of small-volume samples. Such a capability is ideal for use in conjunction with microfluidic mixers, where time resolution may be determined by beam size and sample volumes are kept small to facilitate mixing and conserve material.

Elucidating Internucleosome Interactions and the Roles of Histone Tails

The nucleosome is the first level of genome organization and regulation in eukaryotes where negatively charged DNA is wrapped around largely positively charged histone proteins. Interaction between nucleosomes is dominated by electrostatics at long range and guided by specific contacts at short range, particularly involving their flexible histone tails. We have thus quantified how internucleosome interactions are modulated by salts (KCl, MgCl2) and histone tail deletions (H3, H4 N-terminal), using small-angle x-ray scattering and theoretical modeling. We found that measured effective charges at low salts are ∼1/5th of the theoretically predicted renormalized charges and that H4 tail deletion suppresses the attraction at high salts to a larger extent than H3 tail deletion.

Differential Uptake of Gold Nanoparticles by 2 Species of Tadpole, the Wood Frog (Lithobates Sylvaticus) and the Bullfrog (Lithobates Catesbeianus)

Engineered nanoparticles are aquatic contaminants of emerging concern that exert ecotoxicological effects on a wide variety of organisms. We exposed cetyltrimethylammonium bromide-capped spherical gold nanoparticles to wood frog and bullfrog tadpoles with conspecifics and in combination with the other species continuously for 21 d, then measured uptake and localization of gold. Wood frog tadpoles alone and in combination with bullfrog tadpoles took up significantly more gold than bullfrogs. Bullfrog tadpoles in combination with wood frogs took up significantly more gold than controls. The rank order of weight-normalized gold uptake was wood frogs in combination > wood frogs alone > bullfrogs in combination > bullfrogs alone > controls. In all gold-exposed groups of tadpoles, gold was concentrated in the anterior region compared with the posterior region of the body. The concentration of gold nanoparticles in the anterior region of wood frogs both alone and in combination with bullfrogs was significantly higher than the corresponding posterior regions. We also measured depuration time of gold in wood frogs. After 21 d in a solution of gold nanoparticles, tadpoles lost >83% of internalized gold when placed in gold-free water for 5 d. After 10 d in gold-free water, tadpoles lost 94% of their gold. After 15 d, gold concentrations were below the level of detection. Our finding of differential uptake between closely related species living in similar habitats with overlapping geographical distributions argues against generalizing toxicological effects of nanoparticles for a large group of organisms based on measurements in only one species. Environ Toxicol Chem 2017;36:3351-3358.

Determination of the Composition of the Ion Atmosphere of Condensed DNA Utilizing Inductively-Couple Plasma Atomic Emission Spectroscopy

The problem of reentrant DNA condensation has been studied for decades. Yet despite the immense amount of theoretical and experimental work on this problem, a definitive, experimentally verified model of condensation remains elusive. Using inductively-coupled plasma atomic emission spectroscopy, we have measured the ion composition of condensed DNA under a variety of solution conditions. We have studied not only the ion atmosphere in the condensed DNA, but also how the osmotic pressure and the presence of competing, non-condensing ions affect the final condensed ion atmosphere. These data provide a strong basis against which to measure the various theories of condensation.