p53 Specifically Binds Triplex DNA In Vitro and in Cells.

Triplex DNA is implicated in a wide range of biological activities, including regulation of gene expression and genomic instability leading to cancer. The tumor suppressor p53 is a central regulator of cell fate in response to different type of insults. Sequence and structure specific modes of DNA recognition are core attributes of the p53 protein. The focus of this work is the structure-specific binding of p53 to DNA containing triplex-forming sequences in vitro and in cells and the effect on p53-driven transcription. This is the first DNA binding study of full-length p53 and its deletion variants to both intermolecular and intramolecular T.A.T triplexes. We demonstrate that the interaction of p53 with intermolecular T.A.T triplex is comparable to the recognition of CTG-hairpin non-B DNA structure. Using deletion mutants we determined the C-terminal DNA binding domain of p53 to be crucial for triplex recognition. Furthermore, strong p53 recognition of intramolecular T.A.T triplexes (H-DNA), stabilized by negative superhelicity in plasmid DNA, was detected by competition and immunoprecipitation experiments, and visualized by AFM. Moreover, chromatin immunoprecipitation revealed p53 binding T.A.T forming sequence in vivo. Enhanced reporter transactivation by p53 on insertion of triplex forming sequence into plasmid with p53 consensus sequence was observed by luciferase reporter assays. In-silico scan of human regulatory regions for the simultaneous presence of both consensus sequence and T.A.T motifs identified a set of candidate p53 target genes and p53-dependent activation of several of them (ABCG5, ENOX1, INSR, MCC, NFAT5) was confirmed by RT-qPCR. Our results show that T.A.T triplex comprises a new class of p53 binding sites targeted by p53 in a DNA structure-dependent mode in vitro and in cells. The contribution of p53 DNA structure-dependent binding to the regulation of transcription is discussed.

Intracellular Delivery of Poorly Soluble Polyphenols: Elucidating the Interplay of Self-Assembling Nanocarriers and Human Chondrocytes.

Increased molecular understanding of multifactorial diseases paves the way for novel therapeutic approaches requiring sophisticated carriers for intracellular delivery of actives. We designed and characterized self-assembling lipid-core nanocapsules for coencapsulation of two poorly soluble natural polyphenols curcumin and resveratrol. The polyphenols were identified as high-potential therapeutic candidates intervening in the intracellular inflammation cascade of chondrocytes during the progress of osteoarthritis. To elucidate the interplay between chondrocytes and nanocapsules and their therapeutic effect, we pursued a complementary analytical approach combining label-free visualization with biological assays. Primary human chondrocytes did not show any adverse effects upon nanocapsule application and coherent anti-Stokes Raman scattering images visualized their intracellular uptake. Further, by systematically blocking different uptake mechanisms, an energy independent uptake into the cells could be identified. Additionally, we tested the therapeutic effect of the polyphenol-loaded carriers on inflamed chondrocytes. Treatment with nanocapsules resulted in a major reduction of nitric oxide levels, a well-known apoptosis trigger during the course of osteoarthritis. For a more profound examination of this protective effect on joint cells, we pursued studies with atomic force microscopy investigations. Significant changes in the cell cytoskeleton as well as prominent dents in the cell membrane upon induced apoptosis were revealed. Interestingly, these effects could not be detected for chondrocytes which were pretreated with the nanocapsules. Overall, besides presenting a sophisticated carrier system for joint application, these results highlight the necessity of establishing combinatorial analytical approaches to elucidate cellular uptake, the interplay of codelivered drugs and their therapeutic effect on the subcellular level.

Atomic force microscopy under controlled conditions reveals structure of C-terminal region of α-synuclein in amyloid fibrils.

Atomic force microscopy (AFM) is widely used to measure morphological and mechanical properties of biological materials at the nanoscale. AFM is able to visualize and measure these properties in different environmental conditions. However, these conditions can influence the results considerably, rendering their interpretation a matter of some subtlety. We demonstrate this by imaging ~10 nm diameter α-synuclein amyloid fibrils, focusing specifically on the structure of the C-terminal part of the protein monomers incorporated into fibrils. Despite these influences leading to variations in fibril heights, we have shown that by maintaining careful control of AFM settings we can quantitatively compare the morphological parameters of fibrils imaged in air or in buffer conditions. From this comparison we were able to deduce the semiflexible character of this C-terminal region. Fibril height differences measured in air and liquid indicate that the C-terminal region collapses onto the fibril core upon drying. The fibril heights decrease upon increasing ion concentration in solution, suggesting that the C-terminal tails collapse into more compact structures as a result of charge screening. Finally, PeakForce QNM measurements show an apparent heterogeneity of C-terminal packing along the fibril length.

Spatially resolved frequency-dependent elasticity measured with pulsed force microscopy and nanoindentation.

Recently several atomic force microscopy (AFM)-based surface property mapping techniques like pulsed force microscopy (PFM), harmonic force microscopy or Peakforce QNM® have been introduced to measure the nano- and micro-mechanical properties of materials. These modes all work at different operating frequencies. However, complex materials are known to display viscoelastic behavior, a combination of solid and fluid-like responses, depending on the frequency at which the sample is probed. In this report, we show that the frequency-dependent mechanical behavior of complex materials, such as polymer blends that are frequently used as calibration samples, is clearly measurable with AFM. Although this frequency-dependent mechanical behavior is an established observation, we demonstrate that the new high frequency mapping techniques enable AFM-based rheology with nanoscale spatial resolution over a much broader frequency range compared to previous AFM-based studies. We further highlight that it is essential to account for the frequency-dependent variation in mechanical properties when using these thin polymer samples as calibration materials for elasticity measurements by high-frequency surface property mapping techniques. These results have significant implications for the accurate interpretation of the nanomechanical properties of polymers or complex biological samples. The calibration sample is composed of a blend of soft and hard polymers, consisting of low-density polyethylene (LDPE) islands in a polystyrene (PS) surrounding, with a stiffness of 0.2 GPa and 2 GPa respectively. The spring constant of the AFM cantilever was selected to match the stiffness of LDPE. From 260 Hz to 1100 Hz the sample was imaged with the PFM method. At low frequencies (0.5-35 Hz), single-point nanoindentation was performed. In addition to the material's stiffness, the relative heights of the LDPE islands (with respect to the PS) were determined as a function of the frequency. At the lower operation frequencies for PFM, the islands exhibited lower heights than when measured with tapping mode at 120 kHz. Both spring constants and heights at the different frequencies clearly show a frequency-dependent behavior.

Force spectroscopy and fluorescence microscopy of dsDNA-YOYO-1 complexes: implications for the structure of dsDNA in the overstretching region.

When individual dsDNA molecules are stretched beyond their B-form contour length, they reveal a structural transition in which the molecule extends 1.7 times its contour length. The nature of this transition is still a subject of debate. In the first model, the DNA helix unwinds and combined with the tilting of the base pairs (which remain intact), results in a stretched form of DNA (also known as S-DNA). In the second model the base pairs break resulting effectively in two single-strands, which is referred to as force-induced melting. Here a combination of optical tweezers force spectroscopy with fluorescence microscopy was used to study the structure of dsDNA in the overstretching regime. When dsDNA was stretched in the presence of 10 nM YOYO-1 an initial increase in total fluorescence intensity of the dye-DNA complex was observed and at an extension where the dsDNA started to overstretch the fluorescence intensity leveled off and ultimately decreased when stretched further into the overstretching region. Simultaneous force spectroscopy and fluorescence polarization microscopy revealed that the orientation of dye molecules did not change significantly in the overstretching region (78.0 degrees +/- 3.2 degrees). These results presented here clearly suggest that, the structure of overstretched dsDNA can be explained accurately by force induced melting.

Interaction of oxazole yellow dyes with DNA studied with hybrid optical tweezers and fluorescence microscopy.

We have integrated single molecule fluorescence microscopy imaging into an optical tweezers set-up and studied the force extension behavior of individual DNA molecules in the presence of various YOYO-1 and YO-PRO-1 concentrations. The fluorescence modality was used to record fluorescent images during the stretching and relaxation cycle. Force extension curves recorded in the presence of either dye did not show the overstretching transition that is characteristic for bare DNA. Using the modified wormlike chain model to curve-fit the force extension data revealed a contour length increase of 6% and 30%, respectively, in the presence of YO-PRO-1 and YOYO-1 at 100 nM. The fluorescence images recorded simultaneously showed that the number of bound dye molecules increased as the DNA molecule was stretched and decreased again as the force on the complex was lowered. The binding constants and binding site sizes for YO-PRO-1 and YOYO-1 were determined as a function of the force. The rate of YO-PRO-1 binding and unbinding was found to be 2 orders of magnitude larger than that for YOYO-1. A kinetic model is proposed to explain this observation.

Sequence-specific physical properties of African green monkey alpha-satellite DNA contribute to centromeric heterochromatin formation.

Satellite DNA, a major component of eukaryotic centromeric heterochromatin, is potentially associated with the processes ensuring the faithful segregation of the genetic material during cell division. Structural properties of alpha-satellite DNA (AS) from African green monkey (AGM) were studied. Atomic force microscopy imaging showed smaller end-to-end distances of AS fragments than would be expected for the persistence length of random sequence DNA. The apparent persistence length of the AS was determined as 35nm. Gel-electrophoresis indicated only a weak contribution of intrinsic curvature to the DNA conformations suggesting an additional contribution of an elevated bending flexibility to the reduced end-to-end distances. Next, the force-extension behavior of the naked AS and in complex with nucleosomes was studied using optical tweezers. The naked AS showed a reduced overstretching transition force (-18% the value determined for random DNA) and higher forces required to straighten the DNA. Finally, reconstituted AS nucleosomes disrupted at significantly higher forces as compared with random DNA nucleosomes which is probably due to structural properties of the AS which stabilize the nucleosomes. The data support that the AS plays a role in the formation of centromeric heterochromatin due to specific structural properties and suggest that a relatively higher mechanical stability of nucleosomes is important in AGM-AS chromatin.

Microscale mechanical properties of single elastic fibers: the role of fibrillin-microfibrils.

Micromechanical properties of single elastic fibers and fibrillin-microfibrils, isolated from equine ligamentum nuchae using chemical and enzymatic methods, were determined with atomic force microscopy (AFM). Young's moduli of single elastic fibers immersed in water, devoid of or containing fibrillin-microfibrils, were determined using bending tests. Bending freely suspended elastic fibers on a micro-channeled substrate by a tip-less AFM cantilever generated a force versus displacement curve from which Young's moduli were calculated. For single elastic fibers, Young's moduli in the range of 0.3-1.5 MPa were determined, values not significantly affected by the presence of fibrillin-microfibrils. To further understand the role of fibrillin-microfibrils in vertebrate elastic fibers, layers of fibrillin-microfibrils were subjected to nano-indentation tests. From the slope of the force versus indentation curves, Young's moduli ranging between 0.56 and 0.74 MPa were calculated. The results suggest that fibrillin-microfibrils are not essential for the mechanical properties of single vertebrate elastic fibers.

Mechanical properties of single electrospun collagen type I fibers.

The mechanical properties of single electrospun collagen fibers were investigated using scanning mode bending tests performed with an AFM. Electrospun collagen fibers with diameters ranging from 100 to 600 nm were successfully produced by electrospinning of an 8% w/v solution of acid soluble collagen in 1,1,1,3,3,3-hexafluoro-2-propanol (HFP). Circular dichroism (CD) spectroscopy showed that 45% of the triple helical structure of collagen molecules was denatured in the electrospun fibers. The electrospun fibers were water soluble and became insoluble after cross-linking with glutaraldehyde vapor for 24h. The bending moduli and shear moduli of both non- and cross-linked single electrospun collagen fibers were determined by scanning mode bending tests after depositing the fibers on glass substrates containing micro-channels. The bending moduli of the electrospun fibers ranged from 1.3 to 7.8 GPa at ambient conditions and ranged from 0.07 to 0.26 MPa when immersed in PBS buffer. As the diameter of the fibrils increased, a decrease in bending modulus was measured clearly indicating mechanical anisotropy of the fiber. Cross-linking of the electrospun fibers with glutaraldehyde vapor increased the shear modulus of the fiber from approximately 30 to approximately 50 MPa at ambient conditions.

Mechanical properties of native and cross-linked type I collagen fibrils.

Micromechanical bending experiments using atomic force microscopy were performed to study the mechanical properties of native and carbodiimide-cross-linked single collagen fibrils. Fibrils obtained from a suspension of insoluble collagen type I isolated from bovine Achilles tendon were deposited on a glass substrate containing microchannels. Force-displacement curves recorded at multiple positions along the collagen fibril were used to assess the bending modulus. By fitting the slope of the force-displacement curves recorded at ambient conditions to a model describing the bending of a rod, bending moduli ranging from 1.0 GPa to 3.9 GPa were determined. From a model for anisotropic materials, the shear modulus of the fibril is calculated to be 33 +/- 2 MPa at ambient conditions. When fibrils are immersed in phosphate-buffered saline, their bending and shear modulus decrease to 0.07-0.17 GPa and 2.9 +/- 0.3 MPa, respectively. The two orders of magnitude lower shear modulus compared with the Young's modulus confirms the mechanical anisotropy of the collagen single fibrils. Cross-linking the collagen fibrils with a water-soluble carbodiimide did not significantly affect the bending modulus. The shear modulus of these fibrils, however, changed to 74 +/- 7 MPa at ambient conditions and to 3.4 +/- 0.2 MPa in phosphate-buffered saline.

Micromechanical bending of single collagen fibrils using atomic force microscopy.

A new micromechanical technique was developed to study the mechanical properties of single collagen fibrils. Single collagen fibrils, the basic components of the collagen fiber, have a characteristic highly organized structure. Fibrils were isolated from collagenous materials and their mechanical properties were studied with atomic force microscopy (AFM). In this study, we determined the Young's modulus of single collagen fibrils at ambient conditions from bending tests after depositing the fibrils on a poly(dimethyl siloxane) (PDMS) substrate containing micro-channels. Force-indentation relationships of freely suspended collagen fibrils were determined by loading them with a tip-less cantilever. From the deflection-piezo displacement curve, force-indentation curves could be deduced. With the assumption that the behavior of collagen fibrils can be described by the linear elastic theory of isotropic materials and that the fibrils are freely supported at the rims, a Young's modulus of 5.4 +/- 1.2 GPa was determined. After cross-linking with glutaraldehyde, the Young's modulus of a single fibril increases to 14.7 +/- 2.7 GPa. When it is assumed that the fibril would be fixed at the ends of the channel the Young's moduli of native and cross-linked collagen fibrils are calculated to be 1.4 +/- 0.3 GPa and 3.8 +/- 0.8 GPa, respectively. The minimum and maximum values determined for native and glutaraldehyde cross-linked collagen fibrils represent the boundaries of the Young's modulus.

Combining optical tweezers and scanning probe microscopy to study DNA-protein interactions.

We present the first results obtained with a new instrument designed and built to study DNA-protein interactions at the single molecule level. This microscope combines optical tweezers with scanning probe microscopy and allows us to locate DNA-binding proteins on a single suspended DNA molecule. A single DNA molecule is stretched taut using the optical tweezers, while a probe is scanned along the molecule. Interaction forces between the probe and the sample are measured with the optical tweezers. The instrument thus enables us to correlate mechanical and functional properties of bound proteins with the tension within the DNA molecule. The typical friction force between a micropipette used as probe and a naked DNA molecule was found to be <1 pN. A 16 micro m DNA molecule with approximately 10-15 digoxygenin (DIG) molecules located over a 90 nm range in the middle of the DNA was used as a model system. By scanning with an antidigoxygenin (alpha-DIG) antibody-coated pipette we were able to localize these sites by exploiting the high binding affinity between this antibody-antigen pair. The estimated experimental resolution assuming an infinitesimally thin and rigid probe and a single alpha-DIG/DIG bond was 15 nm.

Micromechanical testing of individual collagen fibrils.

A novel method based on AFM was used to attach individual collagen fibrils between a glass surface and the AFM tip, to allow force spectroscopy studies of these. The fibrils were deposited on glass substrates that are partly coated with Teflon AF. A modified AFM tip was used to accurately deposit epoxy glue droplets on either end of the collagen fibril that cross the glass-Teflon AF interface, as to such attach it with one end to the glass and the other end to the AFM tip. Single collagen fibrils have been mechanically tested in ambient conditions and were found to behave reversibly up to stresses of 90 MPa. Within this regime a Young's modulus of 2-7 GPa was obtained. In aqueous media, the collagen fibrils could be tested reversibly up to about 15 MPa, revealing Young's moduli ranging from 0.2 to at most 0.8 GPa.

Unexpected binding motifs for subnucleosomal particles revealed by atomic force microscopy.

The structure of individual nucleosomes organized within reconstituted 208-12 arrays at different levels of compaction was examined by tapping mode atomic force microscopy in air and liquid. Reconstitution at lower histone octamer to DNA weight ratios showed an extended beads-on-a-string morphology with less than the expected maximum of 12 nucleosome core particles per array, each particle located in the most favored positioning site. A correlation of the contour lengths of these arrays with the number of observed particles revealed two distinct populations of particles, one with approximately 50 nm of bound DNA and a second population with approximately 25 nm. The measured nucleosome center-to-center distances indicate that this approximately 25 nm is not necessarily symmetrically bound about the dyad axis, but can also correspond to DNA bound from either the entry or exit point of the particle to a location at or close to the dyad axis. An assessment of particle heights suggests that particles wrapping approximately 25 nm of DNA are most likely to be subnucleosomal particles, which lack either one or both H2A-H2B dimers. At a higher reconstitution ratio, folded compact arrays fully populated with 12 nucleosome core particles, were observed. Liquid measurements demonstrated dynamic movements of DNA loops protruding from these folded arrays.

Optical tweezers stretching of chromatin.

Recently significant success has emerged from exciting research involving chromatin stretching using optical tweezers. These experiments, in which a single chromatin fibre is attached by one end to a micron-sized bead held in an optical trap and to a solid surface or second bead via the other end, allows manipulation and force detection at a single-molecule level. Through force-induced stretching of chromatin, mechanical properties, specific intermolecular bond strengths and DNA-protein association and dissociation kinetics have been determined. These studies will be extremely fruitful in terms of understanding the function of chromatin structure and its dynamics within the cell.