Xiangyun Qiu

Both helix topology and counterion distribution contribute to the more effective charge screening in dsRNA compared with dsDNA

The recent discovery of the RNA interference mechanism emphasizes the biological importance of short, isolated, double-stranded (ds) RNA helices and calls for a complete understanding of the biophysical properties of dsRNA. However, most previous studies of the electrostatics of nucleic acid duplexes have focused on DNA. Here, we present a comparative investigation of electrostatic effects in RNA and DNA. Using resonant (anomalous) and non-resonant small-angle X-ray scattering, we characterized the charge screening efficiency and counterion distribution around short (25 bp) dsDNA and RNA molecules of comparable sequence. Consistent with theoretical predictions, we find counterion mediated screening to be more efficient for dsRNA than dsDNA. Furthermore, the topology of the RNA A-form helix alters the spatial distribution of counterions relative to B-form DNA. The experimental results reported here agree well with ion-size-corrected non-linear Poisson–Boltzmann calculations. We propose that differences in electrostatic properties aid in selective recognition of different types of short nucleic acid helices by target binding partners.

Divalent counterion-induced condensation of triple-strand DNA.

Understanding and manipulation of the forces assembling DNA/RNA helices have broad implications for biology, medicine, and physics. One subject of significance is the attractive force between dsDNA mediated by polycations of valence ≥3. Despite extensive studies, the physical origin of the “like-charge attraction” remains unsettled among competing theories. Here we show that triple-strand DNA (tsDNA), a more highly charged helix than dsDNA, is precipitated by alkaline-earth divalent cations that are unable to condense dsDNA. We further show that our observation is general by examining several cations (Mg2+, Ba2+, and Ca2+) and two distinct tsDNA constructs. Cation-condensed tsDNA forms ordered hexagonal arrays that redissolve upon adding monovalent salts. Forces between tsDNA helices, measured by osmotic stress, follow the form of hydration forces observed with condensed dsDNA. Probing a well-defined system of point-like cations and tsDNAs with more evenly spaced helical charges, the counterintuitive observation that the more highly charged tsDNA (vs. dsDNA) is condensed by cations of lower valence provides new insights into theories of polyelectrolytes and the biological and pathological roles of tsDNA. Cations and tsDNAs also hold promise as a model system for future studies of DNA–DNA interactions and electrostatic interactions in general.

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.

DNA Methylation Regulated Nucleosome Dynamics

A strong correlation between nucleosome positioning and DNA methylation patterns has been reported in literature. However, the mechanistic model accounting for the correlation remains elusive. In this study, we evaluated the effects of specific DNA methylation patterns on modulating nucleosome conformation and stability using FRET and SAXS. CpG dinucleotide repeats at 10 bp intervals were found to play different roles in nucleosome stability dependent on their methylation states and their relative nucleosomal locations. An additional (CpG)5 stretch located in the nucleosomal central dyad does not alter the nucleosome conformation, but significant conformational differences were observed between the unmethylated and methylated nucleosomes. These findings suggest that the correlation between nucleosome positioning and DNA methylation patterns can arise from the variations in nucleosome stability dependent on their sequence and epigenetic content. This knowledge will help to reveal the detailed role of DNA methylation in regulating chromatin packaging and gene transcription.

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.

Ion Competition in Condensed DNA Arrays in the Attractive Regime

Physical origin of DNA condensation by multivalent cations remains unsettled. Here, we report quantitative studies of how one DNA-condensing ion (Cobalt(3+) Hexammine, or Co(3+)Hex) and one nonDNA-condensing ion (Mg(2+)) 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 Co(3+)Hex ions, consistent with the deficiencies of nonlinear Poisson-Boltzmann approaches in describing multivalent cations. Upon increasing the concentration of Mg(2+), the Co(3+)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 Mg(2+) and Co(3+)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 Co(3+)Hex. It is also observed that only ∼20% DNA charge neutralization by Co(3+)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.

Solution Scattering and FRET Studies on Nucleosomes Reveal DNA Unwrapping Effects of H3 and H4 Tail Removal

Using a combination of small-angle X-ray scattering (SAXS) and fluorescence resonance energy transfer (FRET) measurements we have determined the role of the H3 and H4 histone tails, independently, in stabilizing the nucleosome DNA terminal ends from unwrapping from the nucleosome core. We have performed solution scattering experiments on recombinant wild-type, H3 and H4 tail-removed mutants and fit all scattering data with predictions from PDB models and compared these experiments to complementary DNA-end FRET experiments. Based on these combined SAXS and FRET studies, we find that while all nucleosomes exhibited DNA unwrapping, the extent of this unwrapping is increased for nucleosomes with the H3 tails removed but, surprisingly, decreased in nucleosomes with the H4 tails removed. Studies of salt concentration effects show a minimum amount of DNA unwrapping for all complexes around 50-100mM of monovalent ions. These data exhibit opposite roles for the positively-charged nucleosome tails, with the ability to decrease access (in the case of the H3 histone) or increase access (in the case of the H4 histone) to the DNA surrounding the nucleosome. In the range of salt concentrations studied (0-200mM KCl), the data point to the H4 tail-removed mutant at physiological (50-100mM) monovalent salt concentration as the mononucleosome with the least amount of DNA unwrapping.

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.

Heat induced capsid disassembly and DNA release of bacteriophage λ.

Successive structural changes of bacteriophage upon heating were characterized with quantitative experimental methods. In the commonly used Tris-Mg buffer, differential scanning calorimetry measurements first established that the protein capsid of phage melts at 87°C and its genomic DNA melts at 91°C. Interestingly, prior to the capsid melting, DNA was found to escape out of the capsid and subject to DNase digestion above 68°C, as concluded from light scattering, UV absorption, and electron microscopy studies. Further investigations indicated distinct temperature-dependent behaviors of the three phage proteins. Around 68°C, disruption of the tail first occurs and leads to the escape of DNA; above the capsid melting temperature of 87°C, the auxiliary protein gpD of the phage head remains soluble in solution and resists centrifugal sedimentation, whereas the major capsid protein gpE is easily precipitated and likely exists as aggregates.