Single Point Attachment Research Articles

Comparison of Four Immobilization Methods for Different Transaminases

Biocatalytic syntheses often require unfavorable conditions, which can adversely affect enzyme stability. Consequently, improving the stability of biocatalysts is needed, and this is often achieved by immobilization. In this study, we aimed to compare the stability of soluble and immobilized transaminases from different species. A cysteine in a consensus sequence was converted to a single aldehyde by the formylglycine-generating enzyme for directed single-point attachment to amine beads. This immobilization was compared to cross-linked enzyme aggregates (CLEAs) and multipoint attachments to glutaraldehyde-functionalized amine- and epoxy-beads. Subsequently, the reactivity and stability (i.e., thermal, storage, and solvent stability) of all soluble and immobilized transaminases were analyzed and compared under different conditions. The effect of immobilization was highly dependent on the type of enzyme, the immobilization strategy, and the application itself, with no superior immobilization technique identified. Immobilization of HAGA-beads often resulted in the highest activities of up to 62 U/g beads, and amine beads were best for the hexameric transaminase from Luminiphilus syltensis. Furthermore, the immobilization of transaminases enabled its reusability for at least 10 cycles, while maintaining full or high activity. Upscaled kinetic resolutions (partially performed in a SpinChemTM reactor) resulted in a high conversion, maintained enantioselectivity, and high product yields, demonstrating their applicability.

Toward a Multivalent Synthetic Oligosaccharide-Based Conjugate Vaccine against Shigella: State-of-the-Art for a Monovalent Prototype and Challenges.

This review focuses on the molecular glycovaccine concept, a promising option to develop a Shigella glycoconjugate vaccine. Subsequent to original developments involving, as main vaccine component, the detoxified Shigella lipopolysaccharide randomly conjugated at multiple sites to a carrier protein, novelty stems from the use of rationally designed, well-defined chemically synthesized oligosaccharide haptens conceived as functional surrogates of the main surface antigen, linked via single-point attachment onto a carrier. The concept and design of such a fine-tuned Shigella glycovaccine are presented by way of SF2a-TT15, a neoglycoprotein featuring a synthetic 15-mer oligosaccharide, which constitutes an original vaccine prototype targeting Shigella flexneri 2a, one of the predominant circulating strains in endemic settings. The clinical testing of SF2a-TT15 is summarized with the first-in-human phase I trial in young healthy adults showing a good safety profile and tolerability, while inducing bactericidal antibodies towards S. flexneri 2a bacteria. The proof-of-concept of this novel approach being established, an ongoing phase IIa clinical study in the nine-month-old infant target population in endemic area was launched, which is also outlined. Lastly, some challenges to move forward this original approach toward a multivalent cost-effective Shigella synthetic glycan conjugate vaccine are introduced.

Scalable production and immunogenicity of a cholera conjugate vaccine

There is a need to develop cholera vaccines that are protective in young children under 5 years of age, which induce long-term immunity, and which can be incorporated into the Expanded Programme of Immunization (EPI) in cholera-endemic countries. The degree of protection afforded by currently available oral cholera vaccines (OCV) to young children is significantly lower than that induced by vaccination of older vaccine recipients. Immune responses that protect against cholera target the O-specific polysaccharide (OSP) of Vibrio cholerae, and young children have poor immunological responses to bacterial polysaccharides, which are T cell independent antigens. To overcome this, we have developed a cholera conjugate vaccine (CCV) containing the OSP of V. cholerae O1, the main cause of endemic and epidemic cholera. Here, we describe production of CCV through a scalable manufacturing process and preclinical evaluation of immunogenicity in the presence and absence of aluminum phosphate (alum) as an adjuvant. The vaccine displays V. cholerae O1 Inaba OSP in sun-burst display via single point attachment of core oligosaccharide to a recombinant tetanus toxoid heavy chain fragment (rTTHc). Two different pilot-scale production batches of non-GMP CCV were manufactured and characterized in terms of physico-chemical properties and immunogenicity. In preclinical testing, the vaccine induced OSP- and lipopolysaccharide (LPS)-specific IgG and IgM responses, vibriocidal responses, memory B cell responses, and protection in a V. cholerae O1 challenge model. The addition of alum to the administered vaccine increased OSP-specific immune responses. These results support evaluation of CCV in humans.

The Design and Preclinical Evaluation of a Single-Label Bimodal Nanobody Tracer for Image-Guided Surgery.

Intraoperative guidance using targeted fluorescent tracers can potentially provide surgeons with real-time feedback on the presence of tumor tissue in resection margins. To overcome the limited depth penetration of fluorescent light, combining fluorescence with SPECT/CT imaging and/or gamma-ray tracing has been proposed. Here, we describe the design and preclinical validation of a novel bimodal nanobody-tracer, labeled using a “multifunctional single attachment point” (MSAP) label, integrating a Cy5 fluorophore and a diethylenetriaminepentaacetic acid (DTPA) chelator into a single structure. After conjugation of the bimodal MSAP to primary amines of the anti-HER2 nanobody 2Rs15d and 111In-labeling of DTPA, the tracer’s characteristics were evaluated in vitro. Subsequently, its biodistribution and tumor targeting were assessed by SPECT/CT and fluorescence imaging over 24 h. Finally, the tracer’s ability to identify small, disseminated tumor lesions was investigated in mice bearing HER2-overexpressing SKOV3.IP1 peritoneal lesions. [111In]In-MSAP.2Rs15d retained its affinity following conjugation and remained stable for 24 h. In vivo SPECT/CT and fluorescence images showed specific uptake in HER2-overexpressing tumors with low background. High tumor-to-muscle ratios were obtained at 1h p.i. and remained 19-fold on SPECT/CT and 3-fold on fluorescence images over 24 h. In the intraperitoneally disseminated model, the tracer allowed detection of larger lesions via nuclear imaging, while fluorescence enabled accurate removal of submillimeter lesions. Bimodal nuclear/fluorescent nanobody-tracers can thus be conveniently designed by conjugation of a single-molecule MSAP-reagent carrying a fluorophore and chelator for radioactive labeling. Such tracers hold promise for clinical applications.

A 6-nm ultra-photostable DNA FluoroCube for fluorescence imaging.

Photobleaching limits extended imaging of fluorescent biological samples. Here, we developed DNA based “FluoroCubes” that are similar in size to the green fluorescent protein (GFP), have single-point attachment to proteins, have a ~54-fold higher photobleaching lifetime and emit ~43-fold more photons than single organic dyes. We demonstrate that DNA FluoroCubes provide outstanding tools for single-molecule imaging, allowing the tracking of single motor proteins for >800 steps with nanometer precision.

SOUND SUPPRESSORS FOR SMALL ARMS WITH AN ADVANCED EXPANSION CAMERA

The paper deals with information about sound suppressors for firearms when an annular expansion chamber and two-point mounting attached to the firearm barrel are used. Those patented designs are examined, as from 1910, when the patent for a sound suppressor with such chief essential features was granted. Designs of patented sound suppressors and mechanisms of their operations presented by the authors of inventions are described. The most-used designs of sound suppressors with an annular expansion chamber of the Reflex Suppressors type (Finland) are considered. The authors presented their designs of sound suppressors with an annular expansion chamber. The disadvantages of such designs are reported, including the authors’ designs. When creating mufflers (devices to reduce the sound level of the shot - PSUZV) shot sound for assault rifles (machine guns), sniper weapons, machine guns using high-energy ammunition, to obtain acceptable values for reducing the sound level of the shot muffler need to increase the volume of the hollow volume . This leads to problems in the operation of the weapon with the silencer and ensure the consistency of the barrel of the weapon and the silencer with a single point attachment to the cut of the barrel. One way to solve this problem is to create a muffler with an optional expansion chamber that covers the outside of the barrel and is gas-dynamically coupled to a traditional expansion chamber that protrudes beyond the barrel section through a permeable partition. The main structural elements of such a muffler - inflatable and overhead expansion chambers, a permeable partition between them and two points of attachment - in the cut of the trunk and, as a rule, at the end of the overhead expansion chamber. Information sources, in which data about the special features of thermogasdynamic processes in sound suppressors were published, are listed. In accordance with the results of their analyses variations needed for the sound suppressor design are validated to improve its characteristics. The sound suppressor design, in which outlined variations were realized, is described and the results of fullscale tests are presented. The conclusion about rationality of using the authors’sound suppressors is made, in particular for highpower ammunition of firearms.

Advances in Cyanine - Amino Acid Conjugates and Peptides for Sensing of DNA, RNA and Protein Structures.

Small molecule spectrophotometric probes for DNA/RNA and proteins are of the utmost importance for diagnostics in biochemical and biomedical research. Both, naturally occurring and synthetic probes, often include peptide sequence responsible for the selectivity toward the particular target; however, commercially available dyes are restricted to single point attachment to the peptide (having one reactive group). Here presented are our recent advances in the development of novel amino acidfluorophore probes, with the unique characteristic of free N- and C-terminus available for incorporation at any peptide backbone position. Intriguingly, already monomeric amino acid-fluorophores showed recognition among various DNA/RNA, whereby steric impact and contribution of halogens is systematically studied. Moreover, some dyes revealed intracellular mitochondria specificity. Further, several hetero-dimeric chromophore systems were prepared, demonstrating that synergistic effect can lead to simultaneous DNA, RNA and protein fluorimetric recognition, combined with enzyme inhibition. Also, homodimeric cyanines equipped with chlorine revealed intriguing DNA/RNA selectivity in respect to well-known parent TOTO and YOYO dyes.

Performance Evaluation of Multi-Interfaced Fast Handoff Scheme for PNEMO Environment

Mobility management is classified into two parts such as location management and handoff management. The earlier one concentrates on location update whereas the later one manages continuous Internet connectivity while the Mobile Router (MR) changes its single point of attachment to the network. Therefore, frequent movement of the MR is one of the significant characteristics in Network Mobility (NEMO) environment. Because, in accordance with the standard Network Mobility Basic Support Protocol (NEMO BSP), the MR utilizes single Interface to attach to the access link. MR requires changing its Care of Address (CoA) when it moves among different wireless access networks. As a result, it can directly influence the performance of the mobility management protocols during inter technology handoff of multi-interfaced MR. This paper proposed a multi-interfaced fast handoff scheme in Proxy NEMO (PNEMO) environment. After that, it represents a comparative analysis between the proposed multi-interfaced scheme, NEMO BSP and the PNEMO scheme respectively. The performance disparities of these schemes are estimated and analyzed via both numerical and simulation approaches. The simulation is performed through NS-3 network simulator. The performance metrics estimated for evaluation are mainly handoff delay and packet loss. It has been perceived that, the proposed scheme performs better compared to the PNEMO scheme and NEMO BSP.DOI: http://dx.doi.org/10.5755/j01.eie.24.5.21848

Sugar-Protein Connectivity Impacts on the Immunogenicity of Site-Selective Salmonella O-Antigen Glycoconjugate Vaccines.

A series of glycoconjugates with defined connectivity were synthesized to investigate the impact of coupling Salmonella typhimurium O-antigen to different amino acids of CRM197 protein carrier. In particular, two novel methods for site-selective glycan conjugation were developed to obtain conjugates with single attachment site on the protein, based on chemical modification of a disulfide bond and pH-controlled transglutaminase-catalyzed modification of lysine, respectively. Importantly, conjugation at the C186-201 bond resulted in significantly higher anti O-antigen bactericidal antibody titers than coupling to K37/39, and in comparable titers to conjugates bearing a larger number of saccharides. This study demonstrates that the conjugation site plays a role in determining the immunogenicity in mice and one single attachment point may be sufficient to induce high levels of bactericidal antibodies.

Sugar–Protein Connectivity Impacts on the Immunogenicity of Site‐Selective Salmonella O‐Antigen Glycoconjugate Vaccines

AbstractA series of glycoconjugates with defined connectivity were synthesized to investigate the impact of coupling Salmonella typhimurium O‐antigen to different amino acids of CRM197 protein carrier. In particular, two novel methods for site‐selective glycan conjugation were developed to obtain conjugates with single attachment site on the protein, based on chemical modification of a disulfide bond and pH‐controlled transglutaminase‐catalyzed modification of lysine, respectively. Importantly, conjugation at the C186‐201 bond resulted in significantly higher anti O‐antigen bactericidal antibody titers than coupling to K37/39, and in comparable titers to conjugates bearing a larger number of saccharides. This study demonstrates that the conjugation site plays a role in determining the immunogenicity in mice and one single attachment point may be sufficient to induce high levels of bactericidal antibodies.

Ion-Channels: Goals for Function-Oriented Synthesis

Ion channels provide a conductance pathway for the passive transport of ions across membranes. These functional molecules perform key tasks in biological systems such as neuronal signaling, muscular control, and sensing. Recently, function-oriented synthesis researchers began to focus on ion channels with the goal of modifying the function of existing ion channels (ion selectivity, gating) or creating new channels with novel functions. Both approaches, ion channel engineering and de novo design, have involved synthetic chemists, biochemists, structural biologists, and neurochemists. Researchers characterize the function of ion channels by measuring their conductance in samples of biological membranes (patch clamp) or artificial membranes (planar lipid bilayers). At the single molecule level, these measurements require special attention to the purity of the sample, a challenge that synthetic chemists should be aware of. Ideally, researchers study the function of channels while also acquiring structural data (X-ray, NMR) to understand and predict how synthetic modifications alter channel function. Long-term oriented researchers would like to apply synthetic ion channels to single molecule sensing and to implantat these synthetic systems in living organisms as tools or for the treatment of channelopathies. In this Account, we discuss our own work on synthetic ion channels and explain the shift of our research focus from a de novo design of oligo-THFs and oligo-THF-amino acids to ion channel engineering. We introduce details about two biological lead structures for ion channel engineering: the gramicidin β(6,3) helix as an example of a channel with a narrow ion conductance pathway and the outer membrane porins (OmpF, OmpG) with their open β-barrel structure. The increase and the reversal of ion selectivity of these systems and the hydrophobic match/mismatch of the channel with the phospholipid bilayer are of particular interest. For engineering ion channels, we need to supplement the single-point attachment of a synthetic modulator with the synthesis of a more challenging two-point attachment. The successful function-oriented synthesis of ion channels will require interdisciplinary efforts that include new electrophysiology techniques, efficient synthesis (peptide/protein/organic), and good structural analysis.

Determination of glycation sites by tandem mass spectrometry in a synthetic lactose-bovine serum albumin conjugate, a vaccine model prepared by dialkyl squarate chemistry.

Neoglycoconjugate vaccines synthesized by the squaric acid spacer method allow single point attachment of the carbohydrate antigen to the protein carrier. However, the localization of the carbohydrate antigen sites of conjugation on the protein carrier has been an elusive task difficult to achieve. Covalent attachment of the lactose antigen to the bovine serum albumin (BSA) was prepared by the squaric acid method using a hapten:BSA ratio of 20:1. Different reaction times were used during the conjugation reaction and two different lactose-BSA glycoconjugate vaccines were obtained. The carbohydrate antigen hapten:BSA ratios of these lactose-BSA glycoconjugate vaccines were determined by MALDI-TOF/RTOF-MS and the glycation sites in the neoglycoconjugates were determined using nano-LC/ESI-QqTOF-MS/MS analysis of the trypsin and GluC V8 digests of the conjugates. We have identified a total of 15 glycation sites located on the BSA lysine residues for the neoglycoconjugate vaccine formed with a hapten:BSA ratio of 5.1:1, However, the tryptic and GluC V8 digests of the hapten-BSA glycoconjugate with a hapten:BSA ratio of 19.0:1 allowed identification of 30 glycation sites located on the BSA. These last results seem to indicate that this conjugation results in formation of various glycoforms. It was observed that the number of identified glycation sites increased when the hapten:BSA ratio of glycoconjugate formation increased, and that the location of the glycation sites appears to be mainly on the outer surface of the BSA carrier molecule which is in line with the assumption that the sterically more accessible lysine residues, namely those located on the outer surface of the BSA, would be conjugated preferentially.

Synthesis and evaluation of a bimodal CXCR4 antagonistic peptide.

The antagonistic Ac-TZ14011 peptide, which binds to the chemokine receptor 4, has been labeled with a multifunctional single attachment point reagent that contains a DTPA chelate and a fluorescent dye with Cy5.5 spectral properties. Flow cytometry and confocal microscopy showed that the bimodal labeled peptide gave a specific receptor binding that is similar to monofunctionalized peptide derivatives. Therefore, the newly developed bimodal peptide derivative can be used in multimodal imaging applications.

Actin Cross-link Assembly and Disassembly Mechanics for α-Actinin and Fascin

Self-assembly of complex structures is commonplace in biology but often poorly understood. In the case of the actin cytoskeleton, a great deal is known about the components that include higher order structures, such as lamellar meshes, filopodial bundles, and stress fibers. Each of these cytoskeletal structures contains actin filaments and cross-linking proteins, but the role of cross-linking proteins in the initial steps of structure formation has not been clearly elucidated. We employ an optical trapping assay to investigate the behaviors of two actin cross-linking proteins, fascin and alpha-actinin, during the first steps of structure assembly. Here, we show that these proteins have distinct binding characteristics that cause them to recognize and cross-link filaments that are arranged with specific geometries. alpha-Actinin is a promiscuous cross-linker, linking filaments over all angles. It retains this flexibility after cross-links are formed, maintaining a connection even when the link is rotated. Conversely, fascin is extremely selective, only cross-linking filaments in a parallel orientation. Surprisingly, bundles formed by either protein are extremely stable, persisting for over 0.5 h in a continuous wash. However, using fluorescence recovery after photobleaching and fluorescence decay experiments, we find that the stable fascin population can be rapidly competed away by free fascin. We present a simple avidity model for this cross-link dissociation behavior. Together, these results place constraints on how cytoskeletal structures assemble, organize, and disassemble in vivo.

Divergent Oriented Synthesis For the Design of Reagents for Protein Conjugation

Instead of using diversity oriented syntheses (DOS) to obtain compounds with biological activities, we employed the DOS method to efficiently obtain multifunctional single attachment point (MSAP) reagents for the conjugation to proteins. Acid insensitive functional groups (chelators, fluorochromes) were attached to Lys-Cys-NH(2) or Lys-Lys-betaAla-Cys-NH(2) peptide scaffolds. After cleavage from solid supports, the modified peptide intermediates were split and further modified by two solution phase, chemoselective reactions employing the single amine and single thiol presented on the intermediates. MSAP-based fluorochrome-chelates were obtained, some possessing a third functional group like a polyethylene glycol (PEG) polymer or "click chemistry" reactive alkynes and azides. The DOS of MSAP reagents permitted the efficient generation of panels of MSAP reagents that can be used to obtain multifunctional proteins with a single modified amino acid (a single attachment point).

A Synthetic Carbohydrate-Protein Conjugate Vaccine Candidate against Shigella flexneri 2a Infection

The protective Ag of Shigella, the Gram-negative enteroinvasive bacterium causing bacillary dysentery, or shigellosis, is its O-specific polysaccharide (O-SP) domain of the LPS, the major bacterial surface component. As an alternative to the development of detoxified LPS-based conjugate vaccines, recent effort was put into the investigation of neoglycoproteins encompassing synthetic oligosaccharides mimicking the protective Ags of the O-SP. We previously reported that when coupled to tetanus toxoid via single point attachment, a synthetic pentadecasaccharide representing three biological repeating units of the O-SP of Shigella flexneri 2a (SF2a), one of the most common Shigella serotypes, elicits a better serum anti-LPS 2a Ab response in mice than shorter synthetic O-SP sequences. In this study, we show that the pentadecasaccharide-induced anti-LPS 2a Abs protect passively administered naive mice from Shigella infection. Therefore, this three repeating units sequence, which is recognized by anti-SF2a sera from infected patients, acts as a functional mimic of the native polysaccharide Ag. Analyses of parameters influencing immunogenicity revealed that an investigational SF2a vaccine displaying a pentadecasaccharide:tetanus toxoid molar loading of 14:1 triggers a high and sustained anti-LPS Ab response, without inducing anti-linker Ab, when administered four times at a dose corresponding to 1 mug of carbohydrate. In addition, the profile of the anti-LPS Ab response, dominated by IgG1 production (Th2-type response), mimics that observed in human upon natural SF2a infection. This synthetic carbohydrate-based conjugate may be a candidate for a SF2a vaccine.

Pathology

Glomerulations in prostatic adenocarcinoma are characterized by dilated glands containing intraluminal cribriform structures with a single point of attachment, resembling a renal glomerulus. On prostate biopsy, glomerulations are exclusively associated with carcinoma and not associated with benign mimickers. However, the Gleason grading of carcinoma with glomerulations on needle biopsy remains controversial. We prospectively collected 45 prostate needle biopsies containing carcinoma with glomeruloid features from our consult files for a 9-month period and examined the association between glomerulations and the presence of concurrent high-grade carcinoma. Glomerulations were overwhelmingly associated with high-grade cancer on the same core, composed of either Gleason pattern 4 (n = 36, 80% of cases) or Gleason pattern 5 (n = 2, 4% of cases). Only a minority of glomerulations were surrounded exclusively by pattern 3 cancer (n = 7, 16% of cases) on the same core. Most of the cases with surrounding pattern 4 cancer were scored as 3 + 4 = 7 (n = 24, 66%), whereas a smaller fraction were scored as 4 + 3 = 7 (n = 9, 26%), and only a minority were 4 + 4 = 8 (n = 3, 9%). In most cases, glomeruloid change was present on the same core as the highest Gleason score carcinoma of the case. None of the pattern 3 cases and only a minority of the pattern 4 cancers had higher Gleason score carcinoma on additional cores (n = 5, 14%). Glomeruloid structures are a rare but diagnostic feature of prostatic carcinoma on needle biopsy. Our data indicate that glomerulations are overwhelmingly associated with concurrent Gleason pattern 4 or higher-grade carcinoma. In several cases, transition could be seen among small glomerulations, large glomeruloid structures, and cribriform pattern 4 cancer. These data suggest that glomerulations represent an early stage of cribriform pattern 4 cancer and, until follow-up data are available, are best graded as Gleason pattern 4.

Gleason grading of prostatic adenocarcinoma with glomeruloid features on needle biopsy

Glomerulations in prostatic adenocarcinoma are characterized by dilated glands containing intraluminal cribriform structures with a single point of attachment, resembling a renal glomerulus. On prostate biopsy, glomerulations are exclusively associated with carcinoma and not associated with benign mimickers. However, the Gleason grading of carcinoma with glomerulations on needle biopsy remains controversial. We prospectively collected 45 prostate needle biopsies containing carcinoma with glomeruloid features from our consult files for a 9-month period and examined the association between glomerulations and the presence of concurrent high-grade carcinoma. Glomerulations were overwhelmingly associated with high-grade cancer on the same core, composed of either Gleason pattern 4 (n = 36, 80% of cases) or Gleason pattern 5 (n = 2, 4% of cases). Only a minority of glomerulations were surrounded exclusively by pattern 3 cancer (n = 7, 16% of cases) on the same core. Most of the cases with surrounding pattern 4 cancer were scored as 3 + 4 = 7 (n = 24, 66%), whereas a smaller fraction were scored as 4 + 3 = 7 (n = 9, 26%), and only a minority were 4 + 4 = 8 (n = 3, 9%). In most cases, glomeruloid change was present on the same core as the highest Gleason score carcinoma of the case. None of the pattern 3 cases and only a minority of the pattern 4 cancers had higher Gleason score carcinoma on additional cores (n = 5, 14%). Glomeruloid structures are a rare but diagnostic feature of prostatic carcinoma on needle biopsy. Our data indicate that glomerulations are overwhelmingly associated with concurrent Gleason pattern 4 or higher-grade carcinoma. In several cases, transition could be seen among small glomerulations, large glomeruloid structures, and cribriform pattern 4 cancer. These data suggest that glomerulations represent an early stage of cribriform pattern 4 cancer and, until follow-up data are available, are best graded as Gleason pattern 4.

Live Cell Surface Labeling with Fluorescent Ag Nanocluster Conjugates†

DNA-encapsulated silver clusters are readily conjugated to proteins and serve as alternatives to organic dyes and semiconductor quantum dots. Stable and bright on the bulk and single molecule levels, Ag nanocluster fluorescence is readily observed when staining live cell surfaces. Being significantly brighter and more photostable than organics and much smaller than quantum dots with a single point of attachment, these nanomaterials offer promising new approaches for bulk and single molecule biolabeling.

Nucleosome Immobilization Strategies for Single‐Pair FRET Microscopy

All genomic transactions in eukaryotes take place in the context of the nucleosome, the basic unit of chromatin, which is responsible for DNA compaction. Overcoming the steric hindrance that nucleosomes present for DNA-processing enzymes requires significant conformational changes. The dynamics of these have been hard to resolve. Single-pair Fluorescence Resonance Energy Transfer (spFRET) microscopy is a powerful technique for observing conformational dynamics of the nucleosome. Nucleosome immobilization allows the extension of observation times to a limit set only by photobleaching, and thus opens the possibility of studying processes occurring on timescales ranging from milliseconds to minutes. It is crucial however, that immobilization itself does not introduce artifacts in the dynamics. Here we report on various nucleosome immobilization strategies, such as single-point attachment to polyethylene glycol (PEG) or surfaces coated with bovine serum albumin (BSA), and confinement in porous agarose or polyacrylamide gels. We compare the immobilization specificity and structural integrity of immobilized nucleosomes. A crosslinked star polyethylene glycol coating performs best with respect to tethering specificity and nucleosome integrity, and enables us to reproduce for the first time bulk nucleosome unwrapping kinetics in single nucleosomes without immobilization artifacts.