Photobodies: Light‐Activatable Single‐Domain Antibody Fragments

Photocaged antibody fragments, termed photobodies, have been developed that are impaired in their antigen‐binding capacity and can be activated by irradiation with UV light (365 nm). This rational design concept builds on the selective photocaging of a single tyrosine in a nanobody (a single‐domain antibody fragment). Tyrosine is a frequently occurring residue in central positions of the paratope region. o‐Nitrobenzyl‐protected tyrosine variants were incorporated into four nanobodies, including examples directed against EGFR and HER2, and photodeprotection restores the native sequence. An anti‐GFP photobody exhibited an at least 10 000‐fold impaired binding affinity before photodeprotection compared with the parent nanobody. A bispecific nanobody–photobody fusion protein was generated to trigger protein heterodimerization by light. Photoactivatable antibodies are expected to become versatile protein reagents and to enable novel approaches in diagnostic and therapeutic applications.     

Differences between G‐Protein‐Stabilized Agonist–GPCR Complexes and their Nanobody‐Stabilized Equivalents

Protein nanobodies have been used successfully as surrogates for unstable G‐proteins in order to crystallize G‐protein‐coupled receptors (GPCRs) in their active states. We used molecular dynamics (MD) simulations, including metadynamics enhanced sampling, to investigate the similarities and differences between GPCR–agonist ternary complexes with the α‐subunits of the appropriate G‐proteins and those with the protein nanobodies (intracellular binding partners, IBPs) used for crystallization. In two of the three receptors considered, the agonist‐binding mode differs significantly between the two alternative ternary complexes. The ternary‐complex model of GPCR activation entails enhancement of ligand binding by bound IBPs: Our results show that IBP‐specific changes can alter the agonist binding modes and thus also the criteria for designing GPCR agonists.     

An Amino‐Acid‐Based Self‐Healing Hydrogel: Modulation of the Self‐Healing Properties by Incorporating Carbon‐Based Nanomaterials

An amino‐acid‐based (11‐(4‐(pyrene‐1‐yl)butanamido)undecanoic acid) self‐repairing hydrogel is reported. The native hydrogel, as well as hybrid hydrogels, have been thoroughly characterized by using various microscopic techniques, including transmission electron microscopy (TEM), atomic force microscopy (AFM), Raman spectroscopy, fluorescence spectroscopy, FTIR spectroscopy, X‐ray diffraction, and by using rheological experiments. The native hydrogel exhibited interesting fluorescence properties, as well as a self‐healing property. Interestingly, the self‐healing, thixotropy, and stiffness of the native hydrogel can be successfully modulated by incorporating carbon‐based nanomaterials, including graphene, pristine single‐walled carbon nanotubes (Pr‐SWCNTs), and both graphene and Pr‐SWCNTs, within the native gel system. The self‐recovery time of the gel was shortened by the inclusion of reduced graphene oxide (RGO), Pr‐SWCNTs, or both RGO and Pr‐SWCNTs. Moreover, hybrid gels that contained RGO and/or Pr‐SWCNTs exhibited interesting semiconducting behavior.     

Enrichment of large‐diameter semiconducting single‐walled carbon nanotubes by a mixed‐extractor strategy

In this work, we report a new mixed‐extractor strategy to improve the sorting yield of large‐diameter semiconducting single‐walled carbon nanotubes (s‐SWCNTs) with high purity. In the new mixed‐extractor strategy, two kinds of conjugated polymers with different rigidity, poly(9,9‐n‐dihexyl‐2,7‐fluorene‐alt‐9‐phenyl‐3,6‐carbazole) (PDFP) and poly(9,9‐dioctylfluorene‐alt‐benzothiadiazole) (P8BT), are used to sort large‐diameter s‐SWCNTs through two simple sonication processes. To our surprise, although PDFP itself shows no selectivity toward s‐SWCNTs, it can greatly enhance the sorting yield of P8BT. Using the PDFP/P8BT mixed‐extractor method, the yield of sorted s‐SWCNTs has been enhanced by 5 times with a purity above 99 % in comparison to that using P8BT single‐extractor method. In addition, the photoluminescence (PL) excitation maps shows that the PDFP/P8BT mixed‐extractor system not only enhances the sorting yield substantially, but also tends to be enrichment of (15,4) SWCNTs with the diameter of 1.36 nm.     

Probing the Chemical Functionalization of Single‐Walled Carbon Nanotubes with Multiple Carbon Ad‐Dimer Defects

Drying‐tube‐shaped single‐walled carbon nanotubes (SWCNTs) with multiple carbon ad‐dimer (CD) defects are obtained from armchair (n,n,m) SWCNTs (n=4, 5, 6, 7, 8; m=7, 13). According to the isolated‐pentagon rule (IPR) the drying‐tube‐shaped SWCNTs are unstable non‐IPR species, and their hydrogenated, fluorinated, and chlorinated derivatives are investigated. Interestingly, chemisorptions of hydrogen, fluorine, and chlorine atoms on the drying tube‐shaped SWCNTs are exothermic processes. Compared to the reaction energies for binding of H, F, and Cl atoms to perfect and Stone–Wales‐defective armchair (5,5) nanotubes, binding of F with the multiply CD defective SWCNTs is stronger than with perfect and Stone–Wales‐defective nanotubes. The reaction energy for per F₂ addition is between 85 and 88 kcal mol^?1 more negative than that per H₂ addition. Electronic structure analysis of their energy gaps shows that the CD defects have a tendency to decrease the energy gap from 1.98–2.52 to 0.80–1.17 eV. After hydrogenation, fluorination, and chlorination, the energy gaps of the drying‐tube‐shaped SWCNTs with multiple CD defects are substantially increased to 1.65–3.85 eV. Furthermore, analyses of thermodynamic stability and nucleus‐independent chemical shifts (NICS) are performed to analyze the stability of these molecules.     

Quantum Defects as a Toolbox for the Covalent Functionalization of Carbon Nanotubes with Peptides and Proteins

Single‐walled carbon nanotubes (SWCNTs) are a 1D nanomaterial that shows fluorescence in the near‐infrared (NIR, >800 nm). In the past, covalent chemistry was less explored to functionalize SWCNTs as it impairs NIR emission. However, certain sp³ defects (quantum defects) in the carbon lattice have emerged that preserve NIR fluorescence and even introduce a new, red‐shifted emission peak. Here, we report on quantum defects, introduced using light‐driven diazonium chemistry, that serve as anchor points for peptides and proteins. We show that maleimide anchors allow conjugation of cysteine‐containing proteins such as a GFP‐binding nanobody. In addition, an Fmoc‐protected phenylalanine defect serves as a starting point for conjugation of visible fluorophores to create multicolor SWCNTs and in situ peptide synthesis directly on the nanotube. Therefore, these quantum defects are a versatile platform to tailor both the nanotube's photophysical properties as well as their surface chemistry.     

Extraction of (9,8) Single‐Walled Carbon Nanotubes by Fluorene‐Based Polymers

Selective polymer wrapping is a promising approach to obtain high‐chiral‐purity single‐walled carbon nanotubes (SWCNTs) needed in technical applications and scientific studies. We showed that among three fluorene‐based polymers with different side‐chain lengths and backbones, poly[(9,9‐dihexylfluorenyl‐2,7‐diyl)‐co‐(9,10‐anthracene)] (PFH‐A) can selectively extract SWCNTs synthesized from the CoSO₄/SiO₂ catalyst, which results in enrichment of 78.3 % (9,8) and 12.2 % (9,7) nanotubes among all semiconducting species. These high‐chiral‐purity SWCNTs may find potential applications in electronics, optoelectronics, and photovoltaics. Furthermore, molecular dynamics simulations suggest that the extraction selectivity of PFH‐A relates to the bending and alignment of its alkyl chains and the twisting of its two aromatic backbone units (biphenyl and anthracene) relative to SWCNTs. The strong π–π interaction between polymers and SWCNTs would increase the extraction yield, but it is not beneficial for chiral selectivity. Our findings suggest that the matching between the curvature of SWCNTs and the flexibility of the polymer side chains and the aromatic backbone units is essential in designing novel polymers for selective extraction of (n,m) species.     

Arginine Side Chains as a Dispersant for Individual Single‐Wall Carbon Nanotubes

Charged peptides and proteins disperse single‐wall carbon nanotubes (SWCNTs) in aqueous solutions. However, little is known about the role of their side chains in their interactions with SWCNTs. Homopolypeptide–SWCNT systems are ideal for investigating the mechanisms of such interactions. In this study, we demonstrate that SWCNTs are individually dispersed by poly‐L ‐arginine (PLA). The debundled SWCNTs exhibited a distinct fluorescence. The dispersibility of SWCNTs with PLA was greater than that of SWCNTs with poly‐L ‐lysine (PLL). Molecular dynamics simulations suggest that the side chains of PLA have stronger interactions with the sidewalls of SWCNTs compared with those of PLL. The guanidinium group at the end of the side chain of an arginine residue plays an important role in the interaction with SWCNTs, likely through hydrophobic, van der Waals, and π–π interactions. PLA can be useful as a tool for the dispersion of SWCNTs and can be used to non‐covalently anchor materials to SWCNTs with strong binding.     

Versatile and Efficient Site‐Specific Protein Functionalization by Tubulin Tyrosine Ligase

A novel chemoenzymatic approach for simple and fast site‐specific protein labeling is reported. Recombinant tubulin tyrosine ligase (TTL) was repurposed to attach various unnatural tyrosine derivatives as small bioorthogonal handles to proteins containing a short tubulin‐derived recognition sequence (Tub‐tag). This novel strategy enables a broad range of high‐yielding and fast chemoselective C‐terminal protein modifications on isolated proteins or in cell lysates for applications in biochemistry, cell biology, and beyond, as demonstrated by the site‐specific labeling of nanobodies, GFP, and ubiquitin.     

Supramolecular Surface Modification and Solubilization of Single‐Walled Carbon Nanotubes with Cyclodextrin Complexation

A novel approach to solubilize single‐walled carbon nanotubes (SWCNTs) in the aqueous phase is described by employing supramolecular surface modification. We use cyclodextrin complexes of synthetic molecules that contain a planar pyrene moiety or a bent, shape‐fitted triptycene moiety as a binding group connected through a spacer to an adamantane moiety that is accommodated in the cyclodextrin cavity. The binding groups attach to the sidewalls of SWCNTs through a π–π stacking interaction to yield a supramolecular system that allows the SWCNTs to dissolve in the aqueous phase through the formed hydrophilic cyclodextrin shell. The black aqueous SWCNT solutions obtained are stable over a period of months. They are characterized through absorbance, static, and time‐resolved fluorescence spectroscopy as well as Raman spectroscopy, TEM, and fluorescence‐decay measurements. Furthermore, the shape‐fitted triptycene‐based system shows a pronounced selectivity for SWCNTs with a diameter of 1.0 nm.     

Capillary electrophoresis of covalently functionalized single‐chirality carbon nanotubes

We demonstrate the separation of chirality‐enriched single‐walled carbon nanotubes (SWCNTs) by degree of surface functionalization using high‐performance CE. Controlled amounts of negatively charged and positively charged functional groups were attached to the sidewall of chirality‐enriched SWCNTs through covalent functionalization using 4‐carboxybenzenediazonium tetrafluoroborate or 4‐diazo‐N,N‐diethylaniline tetrafluoroborate, respectively. Surfactant‐ and pH‐dependent studies confirmed that under conditions that minimized ionic screening effects, separation of these functionalized SWCNTs was strongly dependent on the surface charge density introduced through covalent surface chemistry. For both heterogeneous mixtures and single‐chirality‐enriched samples, covalently functionalized SWCNTs showed substantially increased peak width in electropherogram spectra compared to nonfunctionalized SWCNTs, which can be attributed to a distribution of surface charges along the functionalized nanotubes. Successful separation of functionalized single‐chirality SWCNTs by functional density was confirmed with UV‐Vis‐NIR absorption and Raman scattering spectroscopies of fraction collected samples. These results suggest a high degree of structural heterogeneity in covalently functionalized SWCNTs, even for chirality‐enriched samples, and show the feasibility of applying CE for high‐performance separation of nanomaterials based on differences in surface functional density.     

Poly(γ‐benzyl‐L‐glutamate)‐functionalized single‐walled carbon nanotubes from surface‐initiated ring‐opening polymerizations of N‐carboxylanhydride

Single‐walled carbon nanotubes (SWCNTs) have been functionalized with poly(γ‐benzyl‐L ‐glutamate) (PBLG) by ring‐opening polymerizations of γ‐benzyl‐L ‐glutamic acid‐based N‐carboxylanhydrides (NCA‐BLG) using amino‐functionalized SWCNTs (SWCNT‐NH₂) as initiators. The SWCNT functionalization has been verified by FTIR spectroscopy and transmission electron microscopy. The FTIR study reveals that surface‐attached PBLGs adopt random‐coil conformations in contrast to the physically absorbed or bulk PBLGs, which exhibit α‐helical conformations. Raman spectroscopic analysis reveals a significant alteration of the electronic structure of SWCNTs as a result of PBLG functionalization. The PBLG‐functionalized SWCNTs (SWCNT‐PBLG) exhibit enhanced solubility in DMF. Stable DMF solutions of SWCNT‐PBLG/PBLG with a maximum SWCNTs concentration of 259 mg L^?1 can be readily obtained. SWCNT‐PBLG/PBLG solid composites have been characterized by differential scanning calorimetry, thermogravimetric analysis, wide/small‐angle X‐ray scattering (W/SAXS), scanning electron microscopy, and polarized optical microscopy for their thermal or morphological properties. Microfibers containing SWCNT‐PBLG and PBLG can also be prepared via electrospinning. WAXS characterization reveals that SWCNTs are evenly distributed among PBLG rods in solution and in the solid state where PBLGs form a short‐range nematic phase interspersed with amorphous domains. © 2010 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 48: 2340–2350, 2010     

Tetrazine Carbon Nanotubes for Pretargeted In Vivo “Click‐to‐Release” Bioorthogonal Tumour Imaging

The bioorthogonal inverse‐electron‐demand Diels–Alder (IEDDA) cleavage reaction between tetrazine and trans‐cyclooctene (TCO) is a powerful way to control the release of bioactive agents and imaging probes. In this study, a pretargeted activation strategy using single‐walled carbon nanotubes (SWCNTs) that bear tetrazines (TZ@SWCNTs) and a TCO‐caged molecule was used to deliver active effector molecules. To optimize a turn‐on signal by using in vivo fluorescence imaging, we developed a new fluorogenic near‐infrared probe that can be activated by bioorthogonal chemistry and image tumours in mice by caging hemicyanine with TCO (tHCA). With our pretargeting strategy, we have shown selective doxorubicin prodrug activation and instantaneous fluorescence imaging in living cells. By combining a tHCA probe and a pretargeted bioorthogonal approach, real‐time, non‐invasive tumour visualization with a high target‐to‐background ratio was achieved in a xenograft mice tumour model. The combined advantages of enhanced stability, kinetics and biocompatibility, and the superior pharmacokinetics of tetrazine‐functionalised SWCNTs could allow application of targeted bioorthogonal decaging approaches with minimal off‐site activation of fluorophore/drug.     

Quantitative analysis of a ubiquitin‐dependent substrate using capillary electrophoresis with dual laser‐induced fluorescence

Protein degradation by the ubiquitin‐proteasome system (UPS) affects many biological processes. Inhibition of the proteasome has emerged as a potential therapeutic target for cancer treatment. In this study, we developed a method for monitoring the degradation and accumulation of UPS‐dependent substrates in cells using CE with dual LIF. We used a green fluorescent protein (GFP)‐fusion of the ubiquitin substrate ribophorin 1 (GFP‐RPN1) along with red fluorescent protein (RFP) as an internal control to normalize transfection efficiency. Determination of GFP‐RPN1 and RFP in cell lysates were performed in an untreated capillary (75 μm × 50 cm) and 100 mM Tris‐CHES buffer (pH 9.0) containing 10 mM SDS. GFP‐RPN1 and RFP fluorescence were detected at excitation wavelengths of 488 and 635 nm, and emission wavelengths of 520 and 675 nm, respectively, without any interference or crosstalk. The intensity of GFP‐RPN1 fluorescence was normalized to that of RFP. Additionally, the proposed approach was used successfully to detect the degradation of GFP‐RPN1 and evaluate proteasome inhibitors. These results show that the developed method is effective and promising for rapid and quantitative monitoring of UPS‐dependent substrates compared to the current common methods, such as immunoblotting and pulse chase assays.     

Theoretical investigation of the stability,electronic and magnetic properties of thiolated single‐wall carbon nanotubes

The addition of SH and OH groups to single‐wall carbon nanotubes (SWCNTs) was investigated employing first principles calculations. In the case of the semiconducting (10, 0) SWCNT the SWCNT‐SH binding energy is weak, 2–4 kcal/mol. However, for the metallic (5, 5) SWCNT it is larger, 7–9 kcal/mol. Thus metallic SWCNTs seem to be more reactive to SH than the semiconducting ones. Indeed, the (6, 6) SWCNT is more reactive to SH than the (10, 0) SWCNT, by 2–3 kcal/mol, something that can be explained only considering the electronic structure of the tube, because the (6, 6) has a larger diameter. The binding energies are larger for the addition of the OH group, 25 and 30 kcal/mol for the (10, 0) and (5, 5) SWCNTs, respectively. When a single OH or SH group is attached to the metallic SWCNTs, we observe important changes in the DOS at the Fermi level. However, when multiple SH groups are attached, the changes in the electronic and magnetic properties depend on the position of the SH groups. The small binding energy found for the SH addition indicates that the successful functionalization of SWCNTs with SH, SCH₃, and S(CH₂)_nSH groups is mostly due to the presence of defects created after acid treatment and to a minor extent by the metallic tubes present in the samples. Perfect semiconducting SWCNTs showed very low reactivity against the SH group. © 2008 Wiley Periodicals, Inc. Int J Quantum Chem, 2009     

Multi‐Colored Fibers by Self‐Assembly of DNA,Histone Proteins,and Cationic Conjugated Polymers

The development of biomolecular fiber materials with imaging ability has become more and more useful for biological applications. In this work, cationic conjugated polymers (CCPs) were used to construct inherent fluorescent microfibers with natural biological macromolecules (DNA and histone proteins) through the interfacial polyelectrolyte complexation (IPC) procedure. Isothermal titration microcalorimetry results show that the driving forces for fiber formation are electrostatic and hydrophobic interactions, as well as the release of counterions and bound water molecules. Color‐encoded IPC fibers were also obtained based on the co‐assembly of DNA, histone proteins, and blue‐, green‐, or red‐ (RGB‐) emissive CCPs by tuning the fluorescence resonance energy‐transfer among the CCPs at a single excitation wavelength. The fibers could encapsulate GFP‐coded Escherichia coli BL21, and the expression of GFP proteins was successfully regulated by the external environment of the fibers. These multi‐colored fibers show a great potential in biomedical applications, such as biosensor, delivery, and release of biological molecules and tissue engineering.     

Multi‐Colored Fibers by Self‐Assembly of DNA,Histone Proteins,and Cationic Conjugated Polymers

The development of biomolecular fiber materials with imaging ability has become more and more useful for biological applications. In this work, cationic conjugated polymers (CCPs) were used to construct inherent fluorescent microfibers with natural biological macromolecules (DNA and histone proteins) through the interfacial polyelectrolyte complexation (IPC) procedure. Isothermal titration microcalorimetry results show that the driving forces for fiber formation are electrostatic and hydrophobic interactions, as well as the release of counterions and bound water molecules. Color‐encoded IPC fibers were also obtained based on the co‐assembly of DNA, histone proteins, and blue‐, green‐, or red‐ (RGB‐) emissive CCPs by tuning the fluorescence resonance energy‐transfer among the CCPs at a single excitation wavelength. The fibers could encapsulate GFP‐coded Escherichia coli BL21, and the expression of GFP proteins was successfully regulated by the external environment of the fibers. These multi‐colored fibers show a great potential in biomedical applications, such as biosensor, delivery, and release of biological molecules and tissue engineering.     

Synthesis and characterization of polyethylene chains grafted onto the sidewalls of single‐walled carbon nanotubes via copolymerization

Polyethylene (PE) chains grafted onto the sidewalls of SWCNTs (SWCNT‐g‐PE) were successfully synthesized via ethylene copolymerization with functionalized single‐walled carbon nanotubes (f‐SWCNTs) catalyzed by rac‐(en)(THInd)₂ZrCl₂/MAO. Here f‐SWCNTs, in which α‐alkene groups were chemically linked on the sidewalls of SWCNTs, were synthesized by Prato reaction. The composition and microstructure of SWCNT‐g‐PE were characterized by means of ¹H NMR, Fourier transform infrared spectroscopy (FTIR), Raman spectroscopy, thermogravimetric analyses (TGA), field‐emission scanning electron microscope (FESEM), and transmission electron microscope (TEM). Nanosized cable‐like structure was formed in the SWCNT‐g‐PE, in which the PE formed a tubular shell and several SWCNTs bundles existed as core. The formation of the above morphology in the SWCNT‐g‐PE resulted from successfully grafting of PE chains onto the surface of SWCNTs via copolymerization. The grown PE chains grafted onto the sidewall of the f‐SWCNTs promoted the exfoliation of the mass nanotubes. Comparing with pure PE, the physical mixture of PE/f‐SWCNTs and in situ PE/SWCNTs mixture, thermal stability, and mechanical properties of SWCNT‐g‐PE were higher because of the chemical bonding between the f‐SWCNTs and PE chains. © 2007 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 45: 5459–5469, 2007     

Site‐specific reversible immobilization and purification of His‐tagged protein on poly(2‐acetamidoacrylic acid) hydrogel beads

Ni²⁺‐complexed poly(2‐acetamidoacrylic acid) (PAAA) hydrogel beads were developed for the site‐specific reversible immobilization and purification of the histidine‐tagged green fluorescent protein (His‐tagged GFP). PAAA hydrogel beads were prepared by photopolymerization, and significantly improved mechanical properties of PAAA hydrogel beads were observed in comparison with PAAA hydrogel from our previous study. Confocal laser scanning microscopy was used to determine the binding of His‐tagged GFP to the hydrogel beads in three‐dimensional space. Photoluminescence spectroscopy revealed 89% of binding efficiency of His‐tagged GFP to the Ni²⁺‐PAAA hydrogel beads, 51% of yielding recovery. The maximum binding capacity of His‐tagged GFP was estimated to be 0.45 µg/mg of Ni²⁺‐PAAA hydrogel beads. The recombinant His‐tagged GFP from the soluble fraction of E. coli BL21(DE3) cell lysates was purified with Ni²⁺‐PAAA hydrogel beads. The major advantage of the Ni²⁺‐PAAA hydrogel beads system was simple preparation procedures of producing the matrix, because PAAA hydrogel beads had relatively enhanced mechanical strength than soft hydrogels. Copyright © 2012 John Wiley & Sons, Ltd.