Barbara Saccà

Kinetics of tetramolecular quadruplexes

The melting of tetramolecular DNA or RNA quadruplexes is kinetically irreversible. However, rather than being a hindrance, this kinetic inertia allows us to study association and dissociation processes independently. From a kinetic point of view, the association reaction is fourth order in monomer and the dissociation first order in quadruplex. The association rate constant kon, expressed in M−3·s−1 decreases with increasing temperature, reflecting a negative activation energy (Eon) for the sequences presented here. Association is favored by an increase in monocation concentration. The first-order dissociation process is temperature dependent, with a very positive activation energy Eoff, but nearly ionic strength independent. General rules may be drawn up for various DNA and RNA sequence motifs, involving 3–6 consecutive guanines and 0–5 protruding bases. RNA quadruplexes are more stable than their DNA counterparts as a result of both faster association and slower dissociation. In most cases, no dissociation is found for G-tracts of 5 guanines or more in sodium, 4 guanines or more in potassium. The data collected here allow us to predict the amount of time required for 50% (or 90%) quadruplex formation as a function of strand sequence and concentration, temperature and ionic strength.

Orthogonal Protein Decoration of DNA Origami

Structural DNA nanotechnology 2] and the technique of DNA origami enable the rapid generation of a plethora of complex self-assembled nanostructures. Since DNA molecules themselves display limited chemical, optical, and electronic functionality, it is of utmost importance to devise methods to decorate DNA scaffolds with functional moieties to realize applications in sensing, catalysis, and device fabrication. Protein functionalization is particulary desirable because it allows exploitation of an almost unlimited variety of functional elements which nature has evolved over billions of years. The delicate architecture of proteins has resulted in no generally applicable method being currently available to selectively couple these components on DNA scaffolds, and thus approaches used so far are based on reversible antibody– antigen interactions, 9] aptamer binding, 11] nucleic acid hybridization of DNA-tagged proteins, 13] or predominantly biotin–streptavidin (STV) interactions. We demonstrate here that DNA nanostructures can be site-specifically decorated with several different proteins by using coupling systems orthogonal to the biotin–STV system. In particular, benzylguanine (BG) and chlorohexane (CH) groups incorporated in DNA origami have been used as suicide ligands for the site-specific coupling of fusion proteins containing the self-labeling protein tags O-alkylguanine-DNA-alkyltransferase (hAGT), which is often referred to as “Snap-tag”, or haloalkane dehalogenase, which is also known as “HaloTag”. By using various model proteins we demonstrate the general applicability of this approach for the generation of DNA superstructures that are selectively decorated with multiple different proteins. To realize orthogonal protein immobilization on DNA origami using self-ligating protein tags, we chose the Snap-tag, developed by Johnsson and co-workers, and the commercially available HaloTag system. The respective smallmolecule suicide tags (O-benzylguanine (BG) and 5-chlorohexane (CH)) for both self-labeling protein tags are readily available as amino-reactive N-hydroxysuccinimide (NHS) derivatives (BG-NHS and CH-NHS; Figure 1a). Complete derivatization of alkylamino-modified oligonucleotides was achieved by coupling with 30 molar equivalents of BG-NHS or CH-NHS, as indicated by electrophoretic analysis (Figure 1b). To gain access to fusion proteins bearing the complementary Snapand Halo-protein tags, we constructed expression plasmids by genetic fusion of the genes encoding the protein of interest (POI) and Snap-tag or HaloTag (see the Supporting Information). As model POIs we chose the fluorescent proteins enhanced yellow fluorescent protein (EYFP) and mKate, the enzymes cytochrome C peroxidase (CCP) and esterase 2 from Alicyclobacillus acidocaldarius thermos (EST2), to which the self-labeling tags were fused at the C terminus (POI-Snap or POI-Halo, respectively). In addition, the bispecific Halo-Snap fusion protein “covalin”, a chimera which specifically reacts with both BG and CH, as well as monovalent STV (mSTV), were used in this study. The fusion proteins were overexpressed and purified by conventional procedures (see the Supporting Information). The coupling of BGand CHmodified oligonucleotides to the protein was analyzed by using covalin as the initial model to simplify the electrophoretic characterization. It is shown in Figure 1c that both BGand CH-modified single-stranded DNA (ssDNA) oligonucleotides couple effectively to generate the corresponding DNA–covalin conjugates in nearly quantitative yields. DNA coupling of the aforementioned POI fusions, namely mKateSnap, EST2-SNAP, mKate-Halo, CCP-Halo, and EYFP-Halo occurred in a highly specific manner (Figure 1d), and neither Snap or Halo nor mSTV revealed cross-reactivity for the orthogonal-tagged DNA oligomers. We then used SARSE software to aid in the design of face-shaped DNA origami to demonstrate the selective immobilization of protein on DNA nanostructures. Correct folding of M13mp18 ssDNA through the use of 236 staple strands was analyzed by atomic force microscopy (AFM; details of the sequence design as well as experimental procedures are reported in the Supporting Information). Figure 2a illustrates that the face-shaped DNA origami was obtained in high purity, and high-resolution AFM clearly revealed the proposed ears, neck, and seam features of this structure. As an initial test for protein decoration, we selected 23 staple strands, which were biotinylated to create eyes (2 6 [*] Dr. B. Sacc , Dipl.-Chem. R. Meyer, Dipl.-Biotechnol. M. Erkelenz, M. Sc. K. Kiko, A. Arndt, Dr. H. Schroeder, Dr. K. S. Rabe, Prof. C. M. Niemeyer Technische Universit t Dortmund, Fakult t Chemie Biologisch-Chemische Mikrostrukturtechnik Otto-Hahn Strasse 6, 44227 Dortmund (Germany) Fax: (+ 49)231-755-7082 E-mail: christof.niemeyer@tu-dortmund.de [] These authors contributed equally to this work.

The effect of chemical modifications on the thermal stability of different G-quadruplex-forming oligonucleotides

A systematic study of the thermal and conformational properties of chemically modified G-quadruplexes of different molecularities is reported. The effect of backbone charge and atom size, thymine/uracyl substitution as well as the effect of modification at the ribose 2′-position was analyzed by UV spectroscopy. Additional calorimetric studies were performed on different modified forms of the human telomeric sequence. Determination of the differential spectra allowed more insights into the conformational properties of the oligonucleotides. Lack of negative charge at the phosphate backbone yielded to a general destabilization of the G-quadruplex structure. On the other hand, substitution of thymine with uracyl resulted in a moderate or strong stabilization of the structure. Additional modification at the sugar 2′-position gave rise to different effects depending on the molecularity of the quadruplex. In particular, loss of hydrogen bond capacity at the 2′-position strongly affected the conformation of the G-quadruplex. Altogether, these results demonstrate that the effect of some modifications depends on the sequence context, thus providing helpful information for the use of chemically modified quadruplexes as therapeutic agents or as structural elements of supramolecular complexes.

DNA nanomachines and nanostructures involving quadruplexes

DNA is an attractive component for molecular recognition, because of its self-assembly properties. Its three-dimensional structure can differ markedly from the classical double helix. For example, DNA or RNA strands carrying guanine or cytosine stretches associate into four-stranded structures called G-quadruplexes or i-DNA, respectively. Since 2002, several groups have described nanomachines that take advantage of this structural polymorphism. We first introduce the unusual structures that are involved in these devices (i.e., i-DNA and G-quadruplexes) and then describe the opening and closing steps that allow cycling. A quadruplex-duplex molecular machine is then presented in detail, together with the rules that govern its formation, its opening/closing kinetics and the various technical and physico-chemical parameters that play a role in the efficiency of this device. Finally, we review the few examples of nanostructures that involve quadruplexes.

Functionalization of DNA nanostructures with proteins

Proteins possess intrinsic functionalities, which have been optimized in billions of years of natural evolution. The conjugation of proteins with artificial nucleic acids allows one to further functionalize proteins with a synthetically accessible, physicochemically robust tag, which is addressable in a highly specific manner by Watson-Crick hybridization. The resulting DNA-protein conjugates can be advantageously used in a variety of applications, ranging from biomedical diagnostics to DNA-based nanofabrication. This critical review provides an overview on chemical approaches to the synthesis of DNA-protein conjugates and their applications in biomolecular nanosciences (96 references).

Structural Properties of a Collagenous Heterotrimer that Mimics the Collagenase Cleavage Site of Collagen Type I

Collagens contain sequence- and conformation-dependent epitopes responsible for their digestion by collagenases at specific loci. A synthetic heterotrimer construct containing the collagenase cleavage site of collagen type I was found to mimic perfectly native collagen in terms of selectivity and mode of enzymatic degradation. The NMR conformational analysis of this molecule clearly revealed the presence of two structural domains, i.e. a triple helix spanning the Gly-Pro-Hyp repeats and a less ordered portion corresponding to the collagenase cleavage site where the three chains are aligned in extended conformation with loose interchain contacts. These structural properties allow for additional insights into the very particular mechanism of collagen digestion by collagenases.

Binding and Docking of Synthetic Heterotrimeric Collagen Type IV Peptides with α1β1 Integrin

Collagen type IV, whose major and ubiquitous form consists of one 2 and two 1 chains, 2] forms a network that determines the biomechanical stability and macromolecular organization of the basement membrane and provides a scaffold into which other constituents of the tissue are incorporated . This collagen benzotriazol (HOAT) as activating reagents and the coupling times were extended to 3 ±4 h to avoid incomplete peptide bond formation. After the entire biotin-tagged and farnesylated peptide 23 had been assembled on the polymeric support, the seven Aloc groups present were removed simultaneously by treatment with Pd[PPh3]4 in the presence of piperidine for four hours. Removal of the catalyst was achieved by simple washing, which rendered the troublesome purification of the unmasked oligolysine peptide unnecessary. Finally, fully unmasked lipidated K-Ras peptide 2 was released from the solid support by treatment with 1% TFA in the presence of 2% TES. Under these conditions both O-trityl groups present in 24 were removed as well and the farnesyl group remained unattacked. Purification of the target peptide was readily achieved by means of HPLC on an RP-C18 column to yield the desired biotin-tagged and lipidated oligolysine peptide 2 (Figure 1) in high purity and with 11% overall yield.

Dendritic DNA Building Blocks for Amplified Detection Assays and Biomaterials

DNA branches out: Recent advances in the assembly of dendritic DNA structures enable applications in biosensing of pathogens and the generation of novel pads of DNA hydrogel biomaterials (see scheme, left). These pads are immersed in a cell extract containing RNA polymerase (red), ribosomes (yellow), and other components for in vitro protein biosynthesis, where they can be used as templates for cell‐free protein production.WILEY-VCH

Determinants of amyloid fibril degradation by the PDZ protease HTRA1

Excessive aggregation of proteins has a major impact on cell fate and is a hallmark of amyloid diseases in humans. To resolve insoluble deposits and to maintain protein homeostasis, all cells use dedicated protein disaggregation, protein folding and protein degradation factors. Despite intense recent research, the underlying mechanisms controlling this key metabolic event are not well understood. Here, we analyzed how a single factor, the highly conserved serine protease HTRA1, degrades amyloid fibrils in an ATP-independent manner. This PDZ protease solubilizes protein fibrils and disintegrates the fibrillar core structure, allowing productive interaction of aggregated polypeptides with the active site for rapid degradation. The aggregate burden in a cellular model of cytoplasmic tau aggregation is thus reduced. Mechanistic aspects of ATP-independent proteolysis and its implications in amyloid diseases are discussed.

Human High Temperature Requirement Serine Protease A1 (HTRA1) Degrades Tau Protein Aggregates

Background: Protein quality control proteases degrade damaged proteins and protein fragments. Results: The human serine protease HTRA1 degrades tau aggregates and is induced by its substrates. Conclusion: A member of the widely conserved HtrA family is involved in protein quality control in mammalian cells. Significance: HTRA1 might function as a tau protease in vivo. Protective proteases are key elements of protein quality control pathways that are up-regulated, for example, under various protein folding stresses. These proteases are employed to prevent the accumulation and aggregation of misfolded proteins that can impose severe damage to cells. The high temperature requirement A (HtrA) family of serine proteases has evolved to perform important aspects of ATP-independent protein quality control. So far, however, no HtrA protease is known that degrades protein aggregates. We show here that human HTRA1 degrades aggregated and fibrillar tau, a protein that is critically involved in various neurological disorders. Neuronal cells and patient brains accumulate less tau, neurofibrillary tangles, and neuritic plaques, respectively, when HTRA1 is expressed at elevated levels. Furthermore, HTRA1 mRNA and HTRA1 activity are up-regulated in response to elevated tau concentrations. These data suggest that HTRA1 is performing regulated proteolysis during protein quality control, the implications of which are discussed.