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Bacteriophage T7 single-stranded DNA-binding protein (gp2.5) binds to and protects transiently exposed regions of single-stranded DNA (ssDNA) while dynamically interacting with other proteins of the replication complex. We directly visualize fluorescently labelled T7 gp2.5 binding to ssDNA at the single-molecule level. Upon binding, T7 gp2.5 reduces the contour length of ssDNA by stacking nucleotides in a force-dependent manner, suggesting T7 gp2.5 suppresses the formation of secondary structure. Next, we investigate the binding dynamics of T7 gp2.5 and a deletion mutant lacking 21 C-terminal residues (gp2.5-Δ21C) under various template tensions. Our results show that the base sequence of the DNA molecule, ssDNA conformation induced by template tension, and the acidic terminal domain from T7 gp2.5 significantly impact on the DNA binding parameters of T7 gp2.5. Moreover, we uncover a unique template-catalyzed recycling behaviour of T7 gp2.5, resulting in an apparent cooperative binding to ssDNA, facilitating efficient spatial redistribution of T7 gp2.5 during the synthesis of successive Okazaki fragments. Overall, our findings reveal an efficient binding mechanism that prevents the formation of secondary structures by enabling T7 gp2.5 to rapidly rebind to nearby exposed ssDNA regions, during lagging strand DNA synthesis.

Release of growth factors while simultaneously maintaining their full biological activity over a period of days to weeks is an important issue in controlled drug delivery and in tissue engineering. In addition, the selected strategy to immobilize growth factors largely determines their biological activity. Silica surfaces derivatized with glycidyloxy propyl trimethoxysilane and poly(glycidyl methacrylate) brushes yielded epoxide-functionalized surfaces onto which human bone morphogenetic protein-6 (hBMP-6) was immobilized giving stable secondary amine bonds. The biological activity of hBMP-6 was unleashed by hydrolysis of the surface siloxane and ester bonds. We demonstrate that this type of labile bonding strategy can be applied to biomaterial surfaces with relatively simple and biocompatible chemistry, such as siloxane, ester, and imine bonds. Our data indicates that the use of differential hydrolytically labile linkers is a versatile method for functionalization of biomaterials with a variety of growth factors providing control over their biological activity.

We report successful and selective dual protein assembly on patterned bifunctional βCD-Ni(ii)NTA surfaces, using red fluorescent protein variants with hexahistidine-tags and teal fluorescent protein variants conjugated with a peptide containing three adamantyl groups. We show that dual protein patterns can only be assembled, when opposing supramolecular interactions have been optimized and nonspecific interactions have been sufficiently suppressed.

DNA polymerase catalyzes the accurate transfer of genetic information from one generation to the next, and thus it is vitally important for replication to be faithful. DNA polymerase fulfills the strict requirements for fidelity by a combination of mechanisms: 1) high selectivity for correct nucleotide incorporation, 2) a slowing down of the replication rate after misincorporation, and 3) proofreading by excision of misincorporated bases. To elucidate the kinetic interplay between replication and proofreading, we used high-resolution optical tweezers to probe how DNA-duplex stability affects replication by bacteriophage T7 DNA polymerase. Our data show highly irregular replication dynamics, with frequent pauses and direction reversals as the polymerase cycles through the states that govern the mechanochemistry behind high-fidelity T7 DNA replication. We constructed a kinetic model that incorporates both existing biochemical data and the, to our knowledge, novel states we observed. We fit the model directly to the acquired pause-time and run-time distributions. Our findings indicate that the main pathway for error correction is DNA polymerase dissociation-mediated DNA transfer, followed by biased binding into the exonuclease active site. The number of bases removed by this proofreading mechanism is much larger than the number of erroneous bases that would be expected to be incorporated, ensuring a high-fidelity replication of the bacteriophage T7 genome.

We report oriented immobilization of proteins using the standard hexahistidine (His6)-Ni2+:NTA (nitrilotriacetic acid) methodology, which we systematically tuned to give control of surface coverage. Fluorescence microscopy and surface plasmon resonance measurements of self-assembled monolayers (SAMs) of red fluorescent proteins (TagRFP) showed that binding strength increased by 1 order of magnitude for each additional His6-tag on the TagRFP proteins. All TagRFP variants with His6-tags located on only one side of the barrel-shaped protein yielded a 1.5 times higher surface coverage compared to variants with His6-tags on opposite sides of the so-called β-barrel. Time-resolved fluorescence anisotropy measurements supported by polarized infrared spectroscopy verified that the orientation (and thus coverage and functionality) of proteins on surfaces can be controlled by strategic placement of a His6-tag on the protein. Molecular dynamics simulations show how the differently tagged proteins reside at the surface in "end-on" and "side-on" orientations with each His6-tag contributing to binding. Also, not every dihistidine subunit in a given His6-tag forms a full coordination bond with the Ni2+:NTA SAMs, which varied with the position of the His6-tag on the protein. At equal valency but different tag positions on the protein, differences in binding were caused by probing for Ni2+:NTA moieties and by additional electrostatic interactions between different fractions of the β-barrel structure and charged NTA moieties. Potential of mean force calculations indicate there is no specific single-protein interaction mode that provides a clear preferential surface orientation, suggesting that the experimentally measured preference for the end-on orientation is a supra-protein, not a single-protein, effect.

A supramolecular strategy is presented for the assembly of growth factors employing His(6)-tagged single-domain antibodies (VHH). A combination of orthogonal supramolecular interactions of beta-cyclodextrin (beta CD)adamantyl (Ad) hostguest and N-nitrilotriacetic acid (NTA)histidine (His) interactions was employed to generate reversible and homogeneous layers of growth factors. A single-domain antibody VHH fragment was identified to bind to the human bone morphogenetic protein-6 (hBMP6) growth factor and could be recombinantly expressed in E. coli. The VHH fragment was equipped with a C-terminal hexahistidine (His(6)) tether to facilitate the assembly on beta CD surfaces using a linker that contains an Ad group to bind to the beta CD receptors and an NTA moiety to interact with the His(6)-tag upon cocomplexation of Ni2+ ions. After exploring the thermodynamic and kinetic stability of the VHH assemblies on beta CD surfaces using a variety of experimental techniques including microcontact printing (mu CP), surface plasmon resonance (SPR), microscale thermophoresis (MST), and theoretical models for determining the thermodynamic behavior of the system, hBMP6 was assembled onto the VHH-functionalized surfaces. After analyzing the immobilized hBMP6 using immunostaining, the biological activity of hBMP6 was demonstrated in cell differentiation experiments. Early osteogenic differentiation was analyzed in terms of alkaline phosphatase (ALP) activity of KS483-4C3 mouse progenitor cells, and the results indicated that the reversibly immobilized growth factors were functionally delivered to the cells. In conclusion, the supramolecular strategy used here offers the necessary affinity, reversibility, and temporal control to promote biological function of the growth factors that were delivered by this strategy.

The supramolecular assembly of proteins on surfaces has been investigated via the site-selective incorporation of a supramolecular moiety on proteins. To this end, fluorescent proteins have been site-selectively labeled with ferrocenes, as supramolecular guest moieties, via SNAP-tag technology. The assembly of guest-functionalized SNAP-fusion proteins on cyclodextrin- and cucurbit[7]uril-coated surfaces yielded stable monolayers. The binding of all ferrocene fusion proteins is specific as determined by surface plasmon resonance. Micropatterns of the fusion proteins, on patterned cyclodextrin and cucurbituril surfaces, have been visualized using fluorescence microscopy. The SNAP-fusion proteins were also immobilized on cyclodextrin vesicles. The supramolecular SNAP-tag labeling of proteins, thus, allows for the assembly of modified proteins via supramolecular host-guest interaction on different surfaces in a controlled manner. These findings extend the toolbox of fabricating supramolecular protein patterns on surfaces taking advantage of the high labeling efficiency of the SNAP-tag with versatile supramolecular moieties.

Strategies to generate platforms combining tissue targeting and regeneration properties are in great demand in the regenerative medicine field. Here we employ an approach to directly visualize the immobilization of cysteine-terminated peptides on a novel fluorogenic surface. Peptides with relevant biological properties, CLPLGNSH and CLRGRYW, were synthesized to function as peptide binders to transforming growth factor (TGF)-β1 and collagen type II (CII). The selective immobilization of the peptides was directly detected using a fluorogenic surface. Adhered proteins were confined to patterns of these peptides matching with the fluorogenic areas. These results show that the fluorogenic signal can be used to detect the chemo-selective immobilization of non-fluorescent biomolecules and to correlate the cell response with the patterned peptides. After analyzing the sequence specificity and cross-reactivity of the binding of TGF-β1 and CII to the respective peptide regions employing immunofluorescence assays, both peptides were co-immobilized in a step-wise process as detected by the fluorogenic surface. TGF-β1 and CII could be self-sorted from a mixture in a regio-selective manner resulting in a bi-functional protein platform. Surfaces of CLPLGNSH pre-loaded with TGF-β1 showed excellent bioactivity in combination with human articular chondrocytes (HACs) and stimulated expression of chondrogenic markers.

Using poly(trimethylene carbonate) (PTMC) network surfaces containing unreacted macromer methacrylate groups, thiol-functionalized biotin (SH-undecyl-biotin) can be bound to the surface. With micro contact printing techniques and making use of the biotin-streptavidin (SAv) binding system patterned surfaces with biological functionality can be prepared in a very straightforward manner. This method has high potential in numerous biomedical applications.

Using optical tweezers in combination with confocal fluorescence microscopy in a controlled microfluidics environment, results in a robust and versatile methodology to study mechanisms occurring at all levels of the cellular metabolism, up to cell membrane interactions and beyond. Both the high temporal and spatial resolutions that correlative optical tweezers-fluorescence microscopy (CTFM) offers represent an asset to investigate processes with short lifetimes. Additionally, by being able to manipulate and exert tensions to selected molecules, we gain access to important structural features. To demonstrate the potential of this hybrid technique, we performed a series of experiments to study the folding/unfolding dynamics of an intracellular signaling protein and the packaging of DNA within a bacteriophage capsid. Both model systems present transient states with fast kinetics, which would be overlooked using other techniques. Our results show that the applications of this single‑molecule technology are not limited to the field of nucleic acids research and quickly advance to new venues.

Since the first demonstration of employing growth factors (GFs) to control cell behaviour in vitro, the spatiotemporal availability of GFs in vivo has received continuous attention. In particular, the ability to physically confine the mobility of GFs has been used in various tissue engineering applications e.g. stents, orthopaedic implants, sutures and contact lenses. The lack of control over the mobility of GFs in scaffolds jeopardizes their performance in vivo. In this feature article, an overview is given on how to effectively present GFs on scaffolds. In the first part, non-covalent strategies are described covering interaction motifs that are generic to direct GF immobilization. In the second part, covalent strategies are described emphasizing the introduction of reactive groups in existing biomaterials. The feature article ends with a description of strategies based on the physical entrapment of growth factors.

Development of novel methodologies for tethering growth factors (GFs) to materials is highly desired for the generation of biomaterials with improved tissue repair properties. Progress in the development of biomaterials that incorporate GFs and the in vivo performance of such biomaterials in tissue engineering applications, such as stents, orthopaedic implants, sutures and contact lenses, is still challenged by the required control over the mobility of growth factors in biomaterials. Many of the current methodologies to introduce GFs in biomaterials suffer from a lack of control over the spatiotemporal delivery of GFs. The aim of the work described in this thesis is the functional tethering of GFs to biomaterials using reversible chemical strategies with spatiotemporal control, thus following nature’s paradigm. This work consisted of three parts: a) non-covalent strategies have been used to capture GFs to surfaces by employing nanobodies and peptides. In this part of the research considerable attention has been paid as well to fundamental aspects on controlling protein orientation in densely packed layers; b) reversible covalent chemistry has been used to control the spatiotemporal availability of GFs in the extracellular matrix (ECM) by using hydrolysable siloxane and imine bonds as examples and c) a protein array technology has been introduced to create functional platforms of various shapes and content for studying cell behavior. In summary, the reversibility of the tether has been found to play important roles in the biological activity. The results of the studies demonstrate the advantage of tethers that combine immobilized GFs, such as GF stability or the creation of locally highly concentrated GF reservoirs, with released mobile GFs, such as optimization of orientation for an optimal interaction with cellular receptors. Although such systems are attractive, knowledge about the application of such tethering strategies in vivo is limited and deserves detailed attention in future research and leaves ample room for synthesis. For example, stimuli responsive systems might provide tools for a breakthrough in the tissue engineering field.

Adopting supramolecular chemistry for immobilization of proteins is an attractive strategy that entails reversibility and responsiveness to stimuli. The reversible and oriented immobilization and micropatterning of ferrocene-tagged yellow fluorescent proteins (Fc-YFPs) onto β-cyclodextrin (βCD) molecular printboards was characterized using surface plasmon resonance (SPR) spectroscopy and fluorescence microscopy in combination with electrochemistry. The proteins were assembled on the surface through the specific supramolecular host-guest interaction between βCD and ferrocene. Application of a dynamic covalent disulfide lock between two YFP proteins resulted in a switch from monovalent to divalent ferrocene interactions with the βCD surface, yielding a more stable protein immobilization. The SPR titration data for the protein immobilization were fitted to a 1:1 Langmuir-type model, yielding K(LM) = 2.5 × 10(5) M(-1) and K(i,s) = 1.2 × 10(3) M(-1), which compares favorably to the intrinsic binding constant presented in the literature for the monovalent interaction of ferrocene with βCD self-assembled monolayers. In addition, the SPR binding experiments were qualitatively simulated, confirming the binding of Fc-YFP in both divalent and monovalent fashion to the βCD monolayers. The Fc-YFPs could be patterned on βCD surfaces in uniform monolayers, as revealed using fluorescence microscopy and atomic force microscopy measurements. Both fluorescence microscopy imaging and SPR measurements were carried out with the in situ capability to perform cyclic voltammetry and chronoamperometry. These studies emphasize the repetitive desorption and adsorption of the ferrocene-tagged proteins from the βCD surface upon electrochemical oxidation and reduction, respectively.

The overwhelming majority of reactions and interactions in biology do not occur in solution but at interfaces. Therefore, the study of how surfaces play a role in the control of biological interactions poses a great challenge. The interface between synthetic biomaterials and cells, among others, is of particular interest owing to its high impact on the design of novel biomaterials for tissue engineering and biomedical applications. [1] A key aspect of the interfacing of biological material with an artificial substrate is the surface chemistry. This has led to the development of various types of surface chemistry, often in the form of reactive monolayers, thereby allowing the controlled immobilization of biomolecules while paying attention to such properties as control over orientation, retention of biological activity, and selectivity and specificity of the biological interaction. [2] The fabrication of fluorogenic monolayers on surfaces can yield fast, simple, sensitive and nondestructive detection of the immobilization products by fluorescence microscopy. For example, reporter monolayers for immobilization of amine-terminated (bio)molecules [3] for microarrays fabrication [4] and for orthogonal surface modification [5] have been published. This method allows faster and cheaper surface imaging and avoids techniques such as X-ray photoelectron spectroscopy (XPS), time-of-flight secondary ion mass spectrometry (ToF SIMS), and scanning probe microscopy (SPM), while retaining high spatial resolution. The important role that thiols play in nature has encouraged scientists to develop molecular probes for their sensing and quantification in biological systems. [6] For this reason, there are several recent examples of fluorescent and colorimetric sensors for the detection of thiols in solution. However, to the best of our knowledge, no examples exist in the area of surface platforms for biological applications. Developing a platform chemistry for thiols has high potential for reasons such as selectivity and orthogonality of immobilization and the controlled orientation of appropriately bioengineered (e.g., cysteine-modified) peptides and proteins. Maleimide chemistry, because of the high selectivity of its reaction with thiols under physiological conditions, is frequently employed for the selective surface immobilization of biomolecules. [2, 7] Therefore, this reaction is a good candidate for the design of rapid methods to visualize in situ covalent binding of thiols on surfaces, with applications in bioconjugation, bioassays, and materials science. Here we report a strategy for the simultaneous anchoring and detection of molecular and biomolecular thiols in a fast manner, by using a fluorogenic reactive monolayer on glass. This monolayer functions as a molecular construction platform in which the fluorogenic response upon immobilization provides spatial identification and coverage assessment of the thiol immobilization. We show its potential in two applications: colocalization of a dye by supramolecular host–guest chemistry on labeled and pre-immobilized thiol-functionalized cyclodextrin, and colocalization of mouse myoblast cells on fluorescently visualized regions with a pre-immobilized RGD peptide. The fluorogenic probe consists of a coumarin unit equipped with an alkyne moiety that allows its directed immobilization on an azide monolayer on glass by reactive microcontact printing (mCP) with Huisgen 1,3-dipolar cycloaddition. [8] We incorporated a methyl-4-oxo-2-butenoate group next to the coumarin unit (compound 1 in Scheme 1 B) for the binding of thiols by Michael addition. Yi et al. [6a] have recently reported a similar dye for the detection of thiols in solution. The carbon–carbon double bond of the methyl-4-oxo-2-butenoate group quenches the intrinsic fluorescence of the coumarin unit by photoinduced electron transfer (PeT). Upon nucleophilic addition of Scheme 1. A) Functionalization method. Printing of 1 (or 2) by click chemistry is followed by the covalent immobilization and detection of thiols by means of the fluorogenic Michael addition to the methyl-4-oxo-2-butenoate moiety. B) Chemical structures of the compounds employed for the surface immobilization (1 and 2), and the characterization in solution (3).