Mechanistic investigation of smart polymer-protein conjugates

Many affinity separation and diagnostic applications rely upon both capture and release steps. There is thus a need for methods to enhance the reversibility of biomolecular interactions. We have previously demonstrated that stimuli-responsive polymers can be used to gate biomolecular reactions when conjugated near the active site of proteins. Here we have used a new smart polymer, N,N-dimethyl acrylamide-co-4-phenylazophenylacrylate that has allowed a mechanistic investigation of the smart polymer switches. This polymer was conjugated via a vinyl sulfone terminus to cysteine residues of genetically engineered streptavidin mutant E116C, where the polymer is conjugated close to the biotin-binding site, and streptavidin mutant S139C, where the conjugation site is distant. The biotin binding switching activity was strongly dependent on conjugation position, as the E116C conjugate displayed a large thermal response while the S139C conjugate displayed only small effects. Kinetic measurements of biotin release demonstrated that the off-rate of biotin was unperturbed and that the thermally triggered release of biotin with the E116C conjugate was due to the blocking the reassociation of biotin. The addition of free polymer to purified E116C conjugates was also shown to increase the blocking and release properties of the switch. This effect was site dependent, suggesting that the conjugated polymers were directing a physical aggregation near the binding site that effectively enhanced the switching activity. These investigations provide mechanistic insight that can be utilized to design better molecular switches for a variety of stimuli-responsive polymer-protein conjugates.     

Photo-induced unfolding and inactivation of bovine carbonic anhydrase in the presence of a photoresponsive surfactant

Photoreversible changes in the conformation and enzymatic activity of bovine carbonic anhydrase have been investigated as a function of photoresponsive surfactant concentration and light conditions. The light-responsive surfactant undergoes a photoisomerization from the relatively hydrophobic trans isomer under visible light to the relatively hydrophilic cis isomer upon UV illumination, providing a means to photoreversibly control enzyme–surfactant interactions. Small-angle neutron scattering and dynamic light scattering measurements, along with fluorescence spectroscopy, indicate that carbonic anhydrase unfolds upon addition of the surfactant under visible light, while only a small degree of unfolding is observed under UV light. Therefore, the enzyme is completely inactivated in the presence of the trans surfactant, while 40% of the native activity is preserved under UV light, providing a photoreversible “on/off switch” of enzyme activity. Small-angle neutron scattering data provide details of the in vitro conformational changes of the enzyme in response to the photosurfactant and light, with the enzyme found to aggregate as a result of photosurfactant-induced unfolding. Fourier transform infrared (FT-IR) spectroscopy further provides information on the secondary structure changes of the protein in the presence of photosurfactant.     

Photocontrol of kinesin ATPase activity using an azobenzene derivative

Azobenzene is a photochromic molecule that undergoes rapid and reversible isomerization between the cis- and trans-forms in response to ultraviolet (UV) and visible (VIS) light irradiation, respectively. Here, we introduced the sulfhydryl-reactive azobenzene derivative 4-phenylazophenyl maleimide (PAM) into the functional region of kinesin to reversibly regulate the ATPase activity of kinesin by photoirradiation. We prepared five kinesin motor domain mutants, A247C, L249C, A252C, G272C and S275C, which contained a single reactive cysteine residue in loops L11 and L12. These loops are considered to be key regions for the functioning of kinesin as a motor protein. PAM was stoichiometrically incorporated into the cysteine residues in the loops of the mutants. The PAM-modified S275C mutant exhibited reversible alterations in ATPase activity accompanied by cis-trans isomerization upon UV and VIS light irradiation. The ATPase activity exhibited by the cis-isomer of the PAM bound to the mutant was two times higher than that of the trans-isomer. Further, the PAM-modified L249C mutant exhibited reversible alterations in ATPase activity on UV-VIS light irradiation but exhibited the opposite effect on UV and VIS light irradiation. Using a photochromic azobenzene derivative, we have demonstrated that the ATPase activity of the motor protein kinesin is photoregulated.     

In situ control of cell adhesion using photoresponsive culture surface

A photoresponsive culture surface (PRCS) allowing photocontrol of cell adhesion was prepared with a novel polymer material composed of poly(N-isopropylacrylamide) having spiropyran chromophores as side chains. Cell adhesion of the surface was drastically enhanced by the irradiation with ultraviolet (UV) light (wavelength: 365 nm); after subsequent cooling and washing on ice, many cells remained in the irradiated region, whereas most cells were removed from the nonirradiated region. The cell adhesion of the PRCS, which had been enhanced by previous UV irradiation, was reset by the visible light irradiation (wavelength 400-440 nm) and the annealing at 37 degrees C for 2 h. Also it was confirmed that the regional control of cell adhesion was induced several times by repeating the same series of operations. Further, living cell patterning with the 200 microm line width was produced readily by projecting UV light along a micropattern on the PRCS on which the living cells had been seeded uniformly in advance. By using a fluorescent probe that stains living cells only, it was confirmed that the cells maintained sufficient viability even after UV light irradiation followed by cooling and washing.     

Photon-modulated changes of cell attachments on poly(spiropyran-co-methyl methacrylate) membranes

Spiropyran is a photoresponsive molecule, and nonionic spiropyran is reversibly changed by UV irradiation to a hydrophilic polar, zwitterionic merocyanine isomer, and back again by visible light irradiation. A copolymer of nitrobenzospiropyran and methyl methacrylate, poly(NSP-co-MMA) was used as a material with a photosensitive surface. UV irradiation of the photosensitive surface of poly(NSP-co-MMA)-coated glass plates decreased the water contact angles (11 +/- 1 degrees ) and increased diameter of a water drop relative to the unexposed surface. Light-induced detachment of platelets and mesenchymal stem (KUSA-A1) cells on poly(NSP-co-MMA)-coated glass plates was observed upon simple- and patterned-light irradiation, whereas no light-induced detachment of platelets and mesenchymal stem cells was observed on poly(methyl methacrylate)-coated glass plates. This is a result of the change from a closed nonpolar spiropyran to the polar zwitterionic merocyanine isomer induced by UV irradiation. Light-induced detachment of fibrinogen adsorbed on poly(NSP-co-MMA) coated glass plates was also observed in this investigation.     

Thermoprecipitation of streptavidin via oligonucleotide-mediated self-assembly with poly(N-isopropylacrylamide).

A versatile strategy has been developed for selectively and sequentially isolating targets in a liquid-phase affinity separation environment. The strategy uses a recently developed approach for joining together molecules in linkages that are defined by the complementary pairing of oligonucleotides conjugated to the different molecules [Niemeyer, C. M., Sano, T., Smith, C. L., and Cantor, C. R. (1994) Nucleic Acids Res. 22, 5530-9]. In the work presented here, streptavidin was noncovalently coupled with the temperature-responsive poly(N-isopropylacrylamide) [poly(NIPAAM)] through the sequence-specific hybridization of oligonucleotides conjugated to the protein and polymer. A 20-mer oligonucleotide was covalently linked through a heterobifunctional linker to a genetically engineered streptavidin variant that contained a unique cysteine residue at the solvent-accessible site Glu 116. The complementary DNA sequence was conjugated to the end of a linear ester-activated poly(NIPAAM). The two conjugates were allowed to self-assemble in solution via hybridization of their complementary DNA sequences. The streptavidin-poly(NIPAAM) complex could be used to affinity-precipitate radiolabeled biotin or biotinylated alkaline phosphatase above 32 degrees C through the thermally induced phase separation activity of the poly(NIPAAM). The streptavidin-oligo species could then be reversibly separated from the precipitated polymer-oligo conjugate and recycled by lowering the salt concentration, which results in denaturation of the short double-stranded DNA connection. The use of oligonucleotides to couple polymer to streptavidin allows for selective precipitation of different polymers and streptavidin complexes based on the sequence-specific hybridization of their oligonucleotide appendages.     

Light-responsive bioconjugates as novel tools for specific capture of biologicals by photoaffinity precipitation

Light-responsive bioconjugates are synthesized by a two-step protocol calling first for cotelomerization (chain-transfer polymerization) of N-isopropylacrylamide and N-acryloxysuccinimide. The desired bioligand (biotin) is used in modified form as chain-transfer agent in this step. As a consequence, 100% of the produced bioconjugates carry this group. In a second step, the cotelomers (bioconjugates) are rendered photoresponsive by linking a chromophore ((3-aminopropyloxy)azobenzene) group to the N-acryloxysuccinimide side chains. The resulting structures show a critical solution temperature in pure water of 16 degrees C when the azo groups in the side chains are predominately in the (stable) trans state. Irradiation with UV light (330 nm) switches the azo group into the more hydrophilic cis state, and the critical solution temperature rises to 18 degrees C. Irradiation with visible light (> 440 nm) switches the group back to the trans state. Adjusting the temperature to an intermediate level, the bioconjugates are used to demonstrate the concept of photo affinity precipitation, i.e., the specific capture and recovery by light-induced precipitation of a target molecule (avidin) from a serum-containing cell-culture supernatant. The avidin was obtained in highly purified form; no nonspecific copurification of protein impurities was observable.     

Temperature control of biotin binding and release with A streptavidin-poly(N-isopropylacrylamide) site-specific conjugate.

The many laboratory and diagnostic applications utilizing streptavidin as a molecular adaptor rely on its high affinity and essentially irreversible interaction with biotin. However, there are many situations where recovery of the biotinylated molecules is desirable. We have previously shown that poly(N-isopropylacrylamide) (PNIPAAm), a temperature-sensitive polymer, can reversibly block biotin association as the polymer's conformation changes at its lower critical solution temperature (LCST). Here, we have constructed a streptavidin-PNIPAAm conjugate which is able to bind biotin at room temperature or lower and release bound biotin at 37 degrees C. The conjugate can repeatedly bind and release biotin as temperature is cycled through the LCST. A genetically engineered streptavidin mutant, E116C, which has only one cysteine residue, was conjugated site specifically via the sulfhydryl groups with a PNIPAAm that has pendent sulfhydryl-reactive vinyl sulfone groups. The conjugation site is near the tryptophan 120 residue, which forms a van der Waals contact with biotin that is important in generating the large binding free energy. The temperature-induced conformational change of the polymer at position 116 may lead to structural changes in the region of tryptophan 120 that are responsible for the reversible binding between biotin and the conjugated streptavidin.     

Affinity thermoprecipitation and recovery of biotinylated biomolecules via a mutant streptavidin-smart polymer conjugate

A system has been developed for reversibly binding and thermoprecipitating biotinylated macromolecules. A high off-rate Ser45Ala (S45A) streptavidin mutant has been covalently conjugated to poly(N-isopropylacrylamide) (PNIPAAm), a temperature-responsive polymer. The resulting conjugate is shown to coprecipitate biotinylated immunoglobulin G (IgG) and a biotinylated oligonucleotide in response to a thermal stimulus. Thermally precipitated biotinylated macromolecules can be released from the S45A-PNIPAAm conjugate by simple treatment with excess free biotin. This release step has been shown to be unique to the mutant streptavidin conjugate-a conjugate of wild type (WT) streptavidin and PNIPAAm does not release bound biotinylated molecules upon treatment with excess free biotin. The capture efficiency (fraction of target molecule precipitated from solution) of the S45A-PNIPAAm conjugate is similar to that of the WT-PNIPAAm conjugate for the biotinylated IgG target molecule (near 100%), but significantly smaller for the biotinylated oligonucleotide target (approximately 60% for the S45A-PNIPAAm conjugate compared to 80% for the WT-PNIPAAm conjugate). The release efficiency (fraction of originally precipitated target molecule released after treatment with free biotin) of the S45A-PNIPAAm conjugate is 70-80% for the biotinylated IgG target and nears 100% for the biotinylated oligonucleotide target. This system demonstrates the use of a high off-rate streptavidin mutant to add reversibility to a system based on smart-polymer-streptavidin conjugates.     

Photostability of quantum dot micelles under ultraviolet irradiation

Phospholipid quantum dot micelles are useful for bio‐applications because of their amphiphilicity and exceptional biocompatibilities. We investigated the uptake of phospholipid [polyethylene glycol (PEG), biotin, and folic acid terminated] modified CdSe/ZnS quantum dot micelles by cancer cells and its photostability under ultrviolet light in the C spectrum (UV‐C) (254 nm) or UV‐A (365 nm) light irradiation. The stability of micelles to the exposure of UV‐C and UV‐A light was assessed. Biotin‐modified quantum dot micelles give photoluminescence enhancement under UV‐C light irradiation. Folate modified micelle under UV‐C and UV‐A results show considerable photoluminescence enhancement. Photoluminescence lifetime measurements showed 7.04, 8.11 and 11.42 ns for PEG, folate, and biotin terminated phospholipid micelles, respectively. Folate and biotin‐modified quantum dot micelles showed excellent uptake by HeLa cells under fluorescence confocal microscopy. Phospholipid CdSe/ZnS quantum dot micelles can be potentially used for diagnosis and treatment of cancer in the future.     

In vitro selection of a photoresponsive RNA aptamer to hemin

A photoresponsive RNA aptamer to hemin was selected in vitro from a random sequence library of RNAs with azobenzene residues. The aptamer bound to hemin under visible light irradiation and was released by ultraviolet light.     

Phototriggered DNA complexation and compaction using poly(vinyl alcohol) carrying a malachite green moiety

Photoinduced DNA compaction was performed using the interaction of DNA with a photoresponsive random copolymer of poly(vinyl alcohol) carrying a malachite green moiety (PVAMG). Although PVAMG does not have any affinity for DNA under dark conditions, it undergoes photoionization upon exposure to UV light, consequently resulting in a cationic binding site for DNA. Electrophoresis results demonstrated that irradiation of PVAMG retarded the DNA bands due to their complexation, whereas the bands remained unchanged under dark conditions. The binding of PVAMG to DNA occurs at a cationic site/DNA phosphate ratio of approximately 0.036. Single-molecule observations of DNA by fluorescence microscopy revealed that irradiation of PVAMG induced a coil-globule transition in the DNA molecule. Complete compaction of DNA has been accomplished at a cationic site/DNA phosphate ratio >8.0, indicating that PVAMG offers an effective system to photochemically trigger DNA compaction.     

Binding of streptavidin with biotinylated thermosensitive nanospheres based on poly(N,N-diethylacrylamide-co-2-hydroxyethyl methacrylate)

Thermosensitive polymer nanospheres based on N,N-diethylacrylamide and 2-hydroxyethyl methacrylate (HEMA) have been prepared, characterized, and conjugated with biotin. The thermosensitivity of poly(N,N-diethylacrylamide) was enhanced by the incorporation of HEMA up to about 40 mol %. Atomic force microscopic images show that these particles can be closely packed even without the surface charges as in the latex particles. Biotinylation reduces the thermosensitivity of the copolymer nanospheres. The biotinylated hydrogel nanospheres showed a reduction in size upon binding with streptavidin, indicating the formation of a less hydrophilic conjugate. No aggregation of the biotinylated particles due to the cross-linking effect of streptavidin was observed. This size change could be reversed by the addition of free biotin to the system. The interaction is specific, and no such changes were observed when streptavidin was replaced by bovine serum albumin.     

Reversible control of enzyme-inhibitor interactions with light illumination using a photoresponsive surfactant

The effects of a photoresponsive surfactant and light illumination on the complex formed between ribonuclease A (RNase A) and a protein ribonuclease inhibitor (RI) have been investigated to develop a light-based technique to reactivate an enzyme through surfactant-induced dissociation of the enzyme-inhibitor complex. The photoresponsive surfactant undergoes a photoisomerization from the relatively hydrophobic trans isomer under visible light to the relatively hydrophilic cis isomer upon UV illumination, providing a means to reversibly control protein-inhibitor interactions. In the absence of surfactant, RI binds tightly to RNase A through noncovalent interactions, which inhibits the enzyme activity. Upon addition of the surfactant under visible light, RNase A is reactivated, regaining ~75% of the native activity in the absence of RI. In the presence of the surfactant under UV light, however, the enzyme remains inhibited. Fluorescence spectroscopy, dynamic light scattering, and circular dichroism spectroscopy reveal that RI dramatically unfolds upon addition of the trans form of the surfactant, while RNase A does not undergo noticeable structural changes under the same conditions. This indicates that RNase A reactivation occurs through dissociation of the enzyme-inhibitor complex arising from surfactant-induced unfolding of the inhibitor. As a result, photoresponsive surfactant and light illumination can be used as a novel light-based technique to dissociate enzyme-inhibitor complexes and, thus, reactivate an inhibited enzyme.     

Non-radioactive hybridization probes prepared by the chemical labelling of DNA and RNA with a novel reagent, photobiotin.

A photo-activatable analogue of biotin, N-(4-azido-2-nitrophenyl)-N'-(N-d-biotinyl-3-aminopropyl)-N'-methyl-1,3- propanediamine (photobiotin), has been synthesized and used for the rapid and reliable preparation of large amounts of stable, non-radioactive, biotin-labelled DNA and RNA hybridization probes. Upon brief irradiation with visible light, photobiotin formed stable linkages with single- and double-stranded nucleic acids yielding probes which were purified from excess reagent by 2-butanol extraction and ethanol precipitation. Using single-stranded phage M13 DNA probes chemically labelled with one biotin per 100-400 residues and dot-blot hybridization reactions on nitrocellulose, as little as 0.5 pg (6 X 10(-18) mol) of target DNA was detected colorimetrically by avidin or streptavidin complexes with acid or alkaline phosphatase from three commercial sources. The sensitivity of detection of target RNA in dot-blots and Northern blots was equivalent to that obtained with 32p-labelled DNA probes. Photobiotin was also used for the labelling of proteins with biotin.     

Photochemical immobilization of proteins on microwave-synthesized photoreactive polymers

We report a rapid and versatile procedure for the preparation of photoreactive polymers and light-induced immobilization of proteins onto such polymers. Photoreactive controlled-pore glass, silica gel, glass slide, and polystyrene microtiter plate are prepared in 40-60s by microwave irradiation of the respective amino polymers and 1-fluoro-2-nitro-4-azidobenzene. Azido group, now part of the polymer, yields highly reactive nitrene under ultraviolet (UV) light at 365 nm. Thus, when photoreactive polymer and horseradish peroxidase or glucose oxidase are exposed to UV light, the reactive nitrene immobilizes the protein molecules in 10 to 20 min through covalent bonding. As nitrene has a property of inserting into C-H bond, the method may find potential applications for immobilization of biomolecules irrespective of their functional groups.     

Development of Direct Grafting on Cyclic Olefin Copolymers to Improve Hydrophilicity by Using Bioactive Polymers

ObjectivesTo improve the hydrophilicity of cyclic olefin copolymer, a simple and rapid method using two-stage with ultraviolet irradiation was developed in order to graft a bioactive polymer on the surface of these polymers.Materials and MethodsA bioactive polymer, poly(sodium styrene sulfonate) was grafting in two steps on the cyclic olefin copolymer surface. The process consists to activate the surface with ozone and grafting to under UV irradiation in presence of sodium styrene sulfonate. The presence of polymer on the surfaces was characterized by water contact angle, Fourier transform infrared spectroscopy, scanning electron microscopy with energy dispersive spectroscopy and the quantity of polymer grafted was determined by a colorimetric method.ResultsFirst, the time of UV irradiation for the grafting was studied. The results showed that the maximum grafting rate is reached after 60 minutes of reaction. Second, the influence of the presence of additive on the grafting was investigated. The degree of grafting is significantly reduced compared to a sample without additive.ConclusionWe have developed a simple and fast method to graft a hydrophilic and bioactive polymer covalently to a COC surface.     

Light-regulated catalysis by an RNA-cleaving deoxyribozyme

We describe light-induced switches for the catalytic activity of the small, RNA-cleaving 8-17 deoxyribozyme (DNAzyme), based on photochemically induced cis-trans isomerization of azobenzene (Az) moieties covalently tethered at various locations within the DNAzyme. Prior studies have shown that trans-azobenzene is able to stack comfortably within a DNA double helix, stabilizing it, while cis-azobenzene has a helix-destabilizing effect. We designed two classes of Az-modified 8-17DNAzyme constructs, in each of which two azobenzene molecules substituted for nucleotides, either in the substrate-binding arm (SBA); or, within the catalytic core. Measurement of single-turnover kinetics for RNA cleavage revealed that in the SBA constructs Ell and E13, five- to sixfold higher catalytic rates were obtained when the reaction mixture was irradiated with visible light (favouring trans-Az) as compared to ultraviolet light (which promotes cis-Az), consistent with trans-Az in these constructs stabilizing the enzyme-substrate complex. Surprisingly, the reverse result was obtained with the catalytic core construct E17, where ultraviolet irradiation resulted in a five- to sixfold faster catalytic activity relative to visible light irradiation. The development of such light-responsive nucleic acid enzymes may open new possibilities of using light as the activating or repressing agent in the control of gene expression within living cells and organisms.     

Oligonucleotide immobilization on micropatterned streptavidin surfaces

We describe a simple procedure for photolithographic patterning of streptavidin on silicon substrates. Long wavelength UV (365 nm) light was used to direct the covalent attachment of photoactivatable biotin onto silylated silicon wafers. Fluorescently labeled streptavidin was found to bind only in areas exposed to the light. We used this procedure to selectively pattern streptavidin inside microwells etched in silicon, and we investigated the binding characteristics of biotinylated oligonucleotides of lengths, n = 16, 54 and 99 bases. The binding curves were found to fit the functional form of the Langmuir isotherm, with binding saturation proportional to n^–3/4.     

UV light‐tunable fluorescent inks and polymer hydrogel films based on carbon nanodots and lanthanide for enhancing anti‐counterfeiting

Novel water‐soluble green fluorescent carbon nanodots (CNs) using methacrylic acid and m‐phenylenediamine as precursors were first synthesized using a one‐pot hydrothermal method. Red fluorescent lanthanide complexes were prepared using lanthanide ion Eu³⁺ and pyridine‐2,6‐dicarboxylic acid. The optical properties of CNs were characterized using ultraviolet visible (UV) spectra and fluorescence spectra, microscopic morphology was characterized using transmission electron microscopy (TEM) and dynamic light scattering (DLS), and the elemental composition was characterized using Fourier transform‐infrared spectroscopy (FT‐IR) and X‐ray photoelectron spectra (XPS). The fluorescence spectra of the lanthanide complexes were also measured. A simple strategy was developed to prepare UV light‐tunable fluorescent inks and polymer hydrogels films based on CNs and lanthanide complexes. The fluorescent inks and polymer hydrogels films could be repeatedly switched between green and red fluorescence. The change of color depended on luminescence of the CNs and the lanthanide complexes under 254 and 365 nm UV light, respectively. The UV light‐tunable fluorescent inks and polymer hydrogels films could enhance its anti‐counterfeiting function for data and information.