Manipulating the salt and thermal stability of DNA multilayer films via oligonucleotide length

DNA films are promising materials for diverse applications, including sensing, diagnostics, and drug/gene delivery. However, the ability to tune the stability of DNA films remains a crucial aspect for such applications. Herein, we examine the role of oligonucleotide length on the formation, and salt and thermal stability, of DNA multilayer films using oligonucleotides of homopolymeric diblocks (polyAG and polyTC), with each block (A, G, T, or C) ranging from 5 to 30 bases (10-, 20-, 30-, 40-, and 60-mer). Using a combination of quartz crystal microgravimetry, dual polarization interferometry, and flow cytometry, we demonstrate that at least 10 bases per hybridizing block in the DNA diblocks (that is, 20-mer) are required for successful hybridization and, hence, DNA multilayer film formation. Films assembled using longer oligonucleotide blocks were more stable in low salt conditions, with the DNA multilayer films assembled from the 60-mer oligonucleotides remaining intact in solutions of about 25 mM NaCl. A systematic increase in film melting temperature ( T m) was observed for the DNA multilayer films (assembled on colloids) with increasing oligonucleotide length, ranging from 38.5 degrees C for the 20-mer films to 53 degrees C for the 60-mer films. Further, an alternating trend in T m of the DNA multilayer films was observed with layer number (AG or TC); DNA multilayer films terminated with an AG layer exhibited a higher T m (44-49 degrees C) than films with an outermost TC layer (ca. 38 degrees C), suggesting a rearrangement of the film structure upon hybridization of the outermost layer. This work shows that the stability of DNA multilayer films can be tuned by varying the length of the oligonucleotide building blocks, thus providing a versatile means to tailor the salt and thermal stability of DNA films, which is necessary for the application of such films.     

Sequential actions of glucose oxidase and peroxidase in molecular films assembled by layer-by-layer alternate adsorption

Molecular films of protein/polyion layers were assembled by means of alternate adsorption through electrostatic interaction. Glucose oxidase (GOD) and peroxidase (POD) were assembled in combination with sodium poly(styrenesulfonate) (PSS) and poly(ethyleneimine) (PEI), respectively. Enzyme activities of those films on specific substrates (glucose and H(2)O(2)) were examined by coloring reaction of dye DA67. A multienzyme film containing GOD layer and POD layer was prepared by alternate adsorption of POD/PSS followed by PEI/GOD. Sequential redox reaction of glucose/H(2)O(2)/DA67 was demonstrated successfully with this supramolecular system. (c) 1996 John Wiley & Sons, Inc.     

Rational design of cytophilic and cytophobic polyelectrolyte multilayer thin films

Nanostructured polyelectrolyte multilayer thin films electrostatically assembled alternately from such polymers as poly(allylamine hydrochloride) (PAH) and poly(acrylic acid) (PAA) were investigated for their in vitro cell interactions. Not surprisingly, NR6WT cells, a highly adhesive murine fibroblast cell line, attached to many different multilayer combinations tested. However, PAH/PAA multilayers constructed at pH deposition conditions of 2.0/2.0 were completely bioinert. Analogous cell interactions were observed with PAH/poly(methacrylic acid) (PAH/PMA), PAH/sulfonated poly(styrene) (PAH/SPS), and poly(diallyldimethylammonium chloride)/SPS (PDAC/SPS) systems, thereby suggesting a general trend in the fibroblasts' response to multilayers. Specifically, highly ionically stitched films attracted cells, whereas weakly ionically cross-linked multilayers, which swell substantially in physiological conditions to present richly hydrated surfaces, resisted fibroblast attachment. Thus, by manipulating the multilayer pH or ionic strength assembly conditions or both, which in turn dictate the molecular architecture of the thin films, one may powerfully direct a single multilayer combination to be either cell adhesive or cell resistant.     

Cholesterol biosensors prepared by layer-by-layer technique

The analysis of formation, deposition and characterization of cholesterol oxidase (COX) layer-by-layer films were performed. Initially, a layer of polyanion, poly(styrene sulfonate) (PSS) was adsorbed followed by a layer of polycation, poly(ethylene imine) (PEI) on each solid substrate from aqueous solutions. The alternating layers were formed by consecutive adsorption of polycations (PEI) and negatively charged proteins (COX) and cholesterol esterase (CE). A strong interaction between protein and polyelectrolyte improves the stability of the alternating multilayer; however, it can change a native protein conformation and impair the protein activity. The PSS/PEI/COX, PSS/PEI/COX/PEI/CE, PSS/PEI/COX-CE/PEI etc. layered structures were prepared on the surface of a platinum electrode, ITO coated glass plate, quartz crystal microbalance, quartz plates, mica and silicon substrates. Optical and gravimetric measurements based on an ultraviolet–visible absorption spectroscopy and a quartz crystal microbalance revealed that the enzyme multilayers thus prepared consist of molecular layered of the proteins. The surface morphology of such bilayer films was investigated by using atomic force microscopy. The electrochemical redox processes of the enzyme-layered films deposited either on platinum or ITO coated glass plate were investigated. The response current of cholesterol oxidase electrode with concentration of cholesterol was investigated at length.     

Transfection of cells mediated by biodegradable polymer materials with surface-bound polyethyleneimine

Poly(epsilon-CBZ-L-lysine) can be mixed with biodegradable polymers such as poly(D,L-lactic-co-glycolic acid) or poly(L-lactic acid) and formed into films, foams, or microspheres. Surface amino groups may then be deprotected with acid or lithium/liquid ammonia. The amino groups serve as a method to modify the surface by attachment of other molecules. In the present experiments, we show that these polymer materials, as films or foams, may be surface modified by the attachment of polyethyleneimine (PEI). Plasmid DNA attached to the PEI can transfect cells plated on the surface over several days. Covalent atachment of PEI was required for transfection to be efficient. PEI was also attached to surface-bound collagen on cell culture plates and was shown to mediate transfection.     

Covalent layer-by-layer assembly of hyperbranched polyether and polyethyleneimine: multilayer films providing possibilities for surface functionalization and local drug delivery

A convenient and simple route to multifunctional surface coatings via the alternating covalent layer-by-layer (LBL) assembly of p-nitrophenyloxycarbonyl group-terminated hyperbranched polyether (HBPO-NO(2)) and polyethylenimine (PEI) is described. The in situ chemical reaction between HBPO-NO(2) and PEI onto aminolyzed substrates was rapid and mild. Results from ellipsometry measurements, contact angle measurements, and ATR-FTIR spectra confirmed the successful LBL assembly of the building blocks, and the surface reactivity of the multilayer films with HBPO-NO(2) as the outmost layer was demonstrated by the immobilization of an amine-functionalized fluorophore. Furthermore, a biomimetic surface was achieved by surface functionalization of the multilayer films with extracellular matrix protein collagen to promote the adhesion and growth of cells. The studies on the drug loading and in vitro release behaviors of the multilayer films demonstrated their application potentials in local delivery of hydrophilic and hydrophobic therapeutic agents.     

Layer-by-layer assembled carbon nanotubes for selective determination of dopamine in the presence of ascorbic acid

Multilayer films of shortened multi-walled carbon nanotubes (MWNTs) are homogeneously and stably assembled on glassy carbon (GC) electrodes using layer-by-layer (LBL) method based on electrostatic interaction of positively charged poly(diallyldimethylammonium chloride) (PDDA) and negatively charged shortened MWNTs. The assembled MWNT multilayer films were studied with respect to the electrocatalytic activity toward ascorbic acid (AA) and dopamine (DA) and were further applied for selective determination of DA in the presence of AA. Scanning electron microscopy (SEM) used for characterization of MWNT films indicates that the assembled MWNTs are almost in a form of small bundles or single nanotubes on the electrodes. Cyclic voltammetric results with assembled MWNT electrode indicate that the strategy based on the LBL method for assembling the MWNT multilayer films on substrate well retains the electrochemical catalytic activity of the MWNTs toward AA and DA, offering some advantages particularly attractive for analytical applications, such as the form of MWNTs assembled on the substrate, i.e., small bundles or single tubes, homogeneity and stability of the as-assembled MWNT films. These features make the assembled MWNTs relatively potential for selective and sensitive determination of DA in the presence of AA.     

Amperometric glucose biosensor based on layer-by-layer assembly of multilayer films composed of chitosan, gold nanoparticles and glucose oxidase modified Pt electrode

A new strategy for fabricating glucose biosensor was presented by layer-by-layer assembled chitosan (CS)/gold nanoparticles (GNp)/glucose oxidase (GOD) multilayer films modified Pt electrode. First, a cleaned Pt electrode was immersed in poly(allylamine) (PAA), and then transferred to GNp, followed by the adsorption of GOD (GOD/GNp/PAA/Pt). Second, the GOD/GNp/PAA/Pt electrode was immersed in CS, and then transferred to GNp, followed by the adsorption of GOD (GOD/GNp/CS/GOD/GNp/PAA/Pt). Third, different layers of multilayer films modified Pt electrodes were assembled by repeating the second process. Film assembling and characterization were studied by quart crystal microbalance, and properties of the resulting glucose biosensors were measured by electrochemical measurements. The results confirmed that the assembling process of multilayer films was simple to operate, the immobilized GOD displayed an excellent catalytic property to glucose, and GNp in the biosensing interface efficiently improved the electron transfer between analyte and electrode surface. The amperometric response of the biosensors uniformly increased from one to six layers of multilayer films, and then reached saturation after the seven layers. Among the resulting biosensors, the biosensor based on the six layers of multilayer films was best. It showed a wide linear range of 0.5-16 mM, with a detection limit of 7.0 microM estimated at a signal-to-noise ratio of 3, fast response time (within 8s). Moreover, it exhibited good reproducibility, long-term stability and interference free. This method can be used for constructing other thin films, which is a universal immobilization method for biosensor fabrication.     

Bioactive thin film of acidic fibroblast growth factor fabricated by layer-by-layer assembly

A new class of bioactive thin films using growth factors as building blocks has been fabricated via layer-by-layer assembly (LBL) technique. Acid fibroblast growth factor (aFGF) in the presence of heparin was used as negatively charged polyelectrolytes, while poly(ethyleneimine) (PEI) was chosen as a positively charged counterpart. The self-deposition process and surface morphology of the resultant multilayers were monitored and detected by UV-vis absorbance spectra, advanced contact angle measurements, and scanning force microscopy (SFM) observations. Cell culture was performed to assess the efficiency of the growth factors. The fibroblasts proliferated faster on the surface assembled with five bilayers of (aFGF/heparin)/PEI with apparent higher cytoviability than on those surfaces modified by one bilayer of (aFGF/heparin)/PEI, five bilayers of aFGF/PEI, or five bilayers of heparin/PEI, and tissue culture polystyrene. Enhanced secretion of collagen type I and interleukin 6 (IL-6) by the fibroblasts seeded on the five bilayers of (aFGF/heparin)/PEI was also verified by immunohistochemical examination. The bioactivity of the (aFGF/heparin)/PEI multilayers could be largely preserved when stored at -20 degrees C.     

New class of ultrathin,highly cell-adhesion-resistant polyelectrolyte multilayers with micropatterning capabilities

Hydrogen-bonded multilayers comprised of polyacrylamide (PAAm) and a weak polyelectrolyte, such as poly(acrylic acid) (PAA) or poly(methacrylic acid) (PMA), were investigated for their surface-cell interactions. The assembled films were lightly cross-linked thermally or photochemically in order to render them stable in a physiological environment. Both PAA/PAAm and PMA/PAAm multilayers were found to exhibit a high resistance to the adhesion (cytophobicity) of mammalian fibroblasts, even with only a single bilayer coating. Protein adsorption to the multilayers, as revealed by surface plasmon resonance measurements, was greatly reduced for fibronectin and serum-containing medium. In situ swelling experiments indicate that the H-bonded multilayers are hydrogellike coatings capable of a high level of swelling in buffered solution. Utilizing the H-bonding nature of these multilayers, we were able to micropattern the films to create more complex cell-resistant/-adhesive surfaces. The long-term stability of the cell-resistant multilayers was found to be exceptionally good even under conditions (pH 7.4, buffered solution) where a high degree of swelling takes place. No degradation of the micropatterned films was observed over a period of a month, during which time the multilayer coatings remained highly resistant to cell-adhesion.     

Photoelectrochemical, photophysical and morphological studies of electrostatic layer-by-layer thin films based on poly(p-phenylenevinylene) and single-walled carbon nanotubes

The preparation of multilayer films based on poly(p-phenylenevinylene) (PPV) and carboxylic-functionalized single-walled carbon nanotubes (SWNT-COOH) by electrostatic interaction using the layer-by-layer (LbL) deposition method is reported herein. The multilayer build-up, monitored by UV-Vis and photoluminescence (PL) spectroscopies, displayed a linear behavior with the number of PPV and SWNT-COOH layers deposited that undergo deviation and spectral changes for thicker films. Film morphology was evaluated by AFM and epifluorescence microscopies showing remarkable changes after incorporation of SWNT-COOH layers. Films without SWNT show roughness and present dispersed grains; films with SWNT-COOH layers are flatter and some carbon nanotube bundles can be visualized. The photoinduced charge transfer from the conducting polymer to SWNT-COOH was analyzed by PL quenching either by the decrease of the emission intensity or by the presence of dark domains in the epifluorescence micrographs. Photoelectrochemical characterization was performed under white light and the films containing SWNT-COOH displayed photocurrent values between 2.0 μA cm(-2) and 7.5 μA cm(-2), as the amount of these materials increases in the film. No photocurrent was observed for the film without carbon nanotubes. Photocurrent generation was enhanced and became more stable when an intermediate layer of PEDOT:PSS was interposed between the active layer and the ITO electrode, indicating an improvement in hole transfer to the contacts. Our results indicate that these multilayer films are promising candidates as active layers for organic photovoltaic cells.     

Self-assembly effect during the adsorption of polynucleotides on stearic acid langmuir-blodgett monolayer

Interaction of polyadenylic acid, poly(A), with stearic acid Langmuir-Blodgett (LB) monolayer was studied in different electrolyte surroundings. For this purpose LB films of stearic acid, transferred on the mica substrate from poly(A) containing subphase, were analyzed with atomic force microscopy (AFM). The density of polynucleotides surface coverage is ruled by the monovalent electrolyte concentration in the subphase that is in good agreement with previous results. Divalent cations in the subphase are needed to stabilize poly(A) molecules on the surface through formation of "salt bridges". At the very low divalent electrolyte concentration polynucleotides adsorb on the LB film to domains in which the effect of self-assembly is observed. Increase of divalent electrolyte concentration leads to the loss of this orientation effect. The explanation of this effect is proposed.     

Biomacromolecules electrostatic self-assembly on 3-dimensional tissue engineering scaffold

A poly(ethylenimine) (PEI) was employed to obtain a stable positively charged surface on a poly(D,L-lactide) (PDL-LA) tissue engineering scaffold. An extracellular matrix (ECM)-like biomacromolecule, gelatin, was selected as polyelectrolyte and deposit alternately with PEI on the activated PDL-LA scaffold via ESA technique. The zeta-potential result showed alternating charge of polyelectrolytes (PEI/gelatin) layering on PDL-LA microspheres. Quartz crystal microbalance (QCM) measurement further verified the gradual deposition of PEI/gelatin on the PDL-LA thin film. The combination of PEI aminolysis and the layer-by-layer technique was then explored to construct gelatin coating onto the 3-D porous PDL-LA scaffold. Scanning electronic microscopy showed that there is no notable difference between modified and unmodified PLA scaffolds, with regard to the porosity, pore diameter, and scaffold integration. The dual-tunnel confocal laser scanning microscopy indicated uniform gelatin distribution on the inner surface of the 3-D porous scaffold. The gradual build-up of protein layer on scaffold was investigated by radioiodination technique. Chondrocyte was chosen to test the cell behavior on modified and unmodified PDL-LA scaffolds. The results of the cell viability, total intracellular protein content, and cell morphology on the PEI/gelatin multilayers modified PDL-LA scaffold showed to promote chondrocyte growth. Comparing conventional coating methods, polyelectrolyte multilayers are easy and stable to prepare. It may be a promising choice for the surface modification of complex biomedical devices. These very flexible systems allow broad medical applications for drug delivery and tissue engineering.     

Charge-dependent sidedness of cytochrome P450 forms studied by quartz crystal microbalance and atomic force microscopy

Quartz crystal microbalance (QCM) resonance measurements were used to examine the surface charge characteristics of cytochrome P450 forms and the influence of charge on the docking of redox partners like cytochrome b5. The distal surface of cytochrome P450 (CYP)101 (pI = 4.5), relative to the heme, is fairly anionic, as is the proximal surface. The latter, however, also has two cationic clusters. A considerably greater extent of CYP101 binding was seen to the cationic, polyethylene-surfaced resonators. CYP2B4 (pI = 8.5) preferentially bound to the polyanionic, polystyrene sulfonate-surfaced resonators. Cytochrome b5 is an acidic protein that had a preferential binding to the poly(ethyleneimine (PEI)-surfaced resonators. When binding to CYP2B4-surfaced films, cytochrome b5 preferentially bound to those cytochrome P450 molecules that were adsorbed to cationic (PEI) films. It is suggested that adsorption of CYP2B4 to an anionic poly(styrenesulfonate) (PSS) surface is with cationic clusters that include the cytochrome b5 docking domain. This diminishes the extent of docking of the cytochrome b5. In contrast, when CYP2B4 is adsorbed to a cationic film the proximal surface with the cytochrome b5-docking site is available for cytochrome b5 binding. A film of the polycation PEI was adsorbed to the silver QCM surface. It formed polymer islands when viewed with atomic force microscopy. Polyanionic PSS was adsorbed intermittently with the PEI. By the third and fourth layer of polyions the polymer islands were essentially merged and protein adsorption as a fourth or fifth layer formed a nearly continuous film. CYP101 was seen to adsorb as globules with a molecular diameter of about 10 nm. CYP2B4 adsorbed to the polyionic films had a slightly elliptical globular shape, also with a molecular diameter of about 10 nm.     

Electrostatic adsorption of heme proteins alternated with polyamidoamine dendrimers for layer-by-layer assembly of electroactive films

A novel thin film of heme proteins, including hemoglobin (Hb), myoglobin (Mb), and catalase (Cat), was successfully assembled layer by layer with polyamidoamine (PAMAM) dendrimers on different solid surfaces. At pH 7.0, protonated PAMAM possesses positive surface charges, whereas the proteins have net negative surface charges at pH above their isoelectric points. Thus, layer-by-layer {PAMAM/protein}(n)() films were assembled with alternate adsorption of oppositely charged PAMAM and proteins from their aqueous solutions mainly by electrostatic interaction. The assembly process was monitored by quartz crystal microbalance (QCM), UV-vis spectroscopy, and cyclic voltammetry (CV). The growth of the protein multilayer films was regular and linear, whereas the electroactivity of the films was only extended to a few bilayers. CVs of {PAMAM/protein}(n)() films showed a pair of well-defined and nearly reversible peaks characteristic of the protein heme Fe(III)/Fe(II) redox couples. Although {PAMAM/Hb}(n)() and {PAMAM/Mb}(n)() films showed very similar properties, {PAMAM/Cat}(n)() films displayed different and unique characters. The substrates with biological or environmental significance, such as oxygen, hydrogen peroxide, trichloroacetic acid, and nitrite, were catalytically reduced at {PAMAM/protein}(n)() film electrodes, showing the potential applicability of the films as new types of biosensors or bioreactors based on direct electrochemistry of the proteins. Both the electrochemical and electrocatalytic activity of {PAMAM/protein}(n)() films can be tailored precisely by controlling the number of bilayers or the film thickness.     

Polyelectrolyte multilayer films as substrates for photoreceptor cells

Reconstruction of extracellular matrix substrates for delivery of functional photoreceptors is crucial in pathologies such as retinal degeneration and age-related macular degeneration. In this study, we assembled polyelectrolyte films using the layer-by-layer deposition method. The buildup of three different films composed of poly(L-lysine)/chondroitin sulfate (PLL/CSA), poly(L-lysine)/poly(styrenesulfonate) (PLL/PSS), or poly(L-lysine)/hyaluronic acid (PLL/HA) was followed by means of quartz crystal microbalance measurements, optical waveguide light mode spectroscopy, confocal microscopy, and atomic force microscopy. The exponential growth regime and the diffusion of PLL chains from the bulk through the PLL/CSA, PLL/PSS, and PLL/HA films was examined. Evaluation of photoreceptor cell viability was optimal on one layer of PLL (PLL(1)), followed by 10 bilayers of PLL/HA [(PLL/HA)(10)] and 10 bilayers of PLL/CSA [(PLL/CSA)(10)]. The number of bilayers and the type of terminating layer also had a significant influence on the number of photoreceptor cells attached. Functionalized polyelectrolyte multilayer films were obtained by adsorbing basic fibroblastic factor (bFGF) or the insoluble fraction of interphotoreceptor matrix (IPM) on or within polyelectrolyte multilayers. bFGF and IPM adsorption on top of the (PLL/CSA)(10)/PLL polyelectrolyte films increased the number of photoreceptor cells attached and maintained the differentiation of rod and cone cells.     

The location of cytochrome c on the surface of ultrathin lipid multilayer films using x-ray diffraction.

X-ray diffraction and spectroscopic techniques were used to characterize ultrathin fatty acid multilayers having a bound surface layer of cytochrome c. Three to six monolayers of arachidic acid were deposited onto an alkylated glass surface, using the Langmuir-Blodgett method. These fatty acid multilayer films were stored either in a 1 mM NaHCO3 pH 7.5 solution or a buffered 10 microM cytochrome c solution, pH 7.5. After washing extensively with buffer, these multilayer films were assayed for bound cytochrome c by optical spectroscopy. It was found that the cytochrome c bound only to the odd-numbered monolayer films (which have hydrophilic surfaces). The theoretical number of cytochrome c molecules bound to the ultrathin multilayer films having three or five monolayers was calculated as N = 1.2 x 10(13)/cm2 (assuming a hexagonally close-packed monolayer of protein), which would produce an optical density of 0.002 at a wavelength of 550 nm; for a three or five monolayer ultrathin film that was incubated with cytochrome c, OD550 approximately equal to 0.002. The protein was released from the film when treated with greater than 100 mM KCl solution, as would be expected for an electrostatic interaction. Meridional x-ray diffraction data were collected from the arachidic acid films with and without a bound cytochrome c layer. A box refinement technique, previously shown to be effective in deriving the profile structures of nonperiodic ultrathin films, was used to determine the multilayer electron density profiles. The electron density profiles and their autocorrelation functions showed that bound cytochrome c resulted in an additional electron dense feature on the multilayer surface, consistent with a bound cytochrome c monolayer. The position of the bound protein relative to the multilayer surface was independent of the number of fatty acid monolayers in the multilayer. Future studies will use these methods to investigate the structures of membrane protein complexes bound directly to the surface of multilayer films.     

Investigation of selective protein immobilization on charged protein array by wavelength interrogation-based SPR sensor

We have investigated the use of multilayer films of polyelectrolytes as selective surfaces to analyze protein interactions with a self-assembled SPR wavelength-shift sensor. Charged arrays were prepared by alternating adsorption of the charged polyelectrolytes, poly(diallyldimethylammonium chloride) (PDDA) and poly(sodium 4-styrenesulfonate) (PSS). Multilayer formation was monitored with the SPR wavelength-shift sensor and a Spreeta SPR sensor. Protein immobilization on the charged surfaces, which was also analyzed by the SPR sensors, was dependent on the pI of the proteins. Tissue transglutaminase (tTGase) and beta-galactosidase (pIs, 5.1 and 5.3, respectively) were preferentially bound to the positively charged PDDA surface, whereas lysozyme (pI, 11.0) was selectively bound to the negatively charged PSS surface. Immobilization of tTGase on the PDDA surface was also dependent on the buffer pH. The interaction of tTGase with RhoA(V14), a constitutively active form of Rho, could be detected on the charged arrays with the wavelength-shift sensor. The arrays could be reutilized at least 5 times. Thus, it is likely that charged surfaces, assembled by the layer-by-layer method using polyelectrolytes, will prove useful for preparing selective protein arrays.     

Dependence of 4'-(hydroxymethyl)-4,5',8-trimethylpsoralen photoaddition on the conformation of ribonucleic acid

The photoaddition of 4'-(hydroxymethyl)-4,5',8-trimethylpsoralen (HMT) to different conformational states of RNA was studied. Poly(U), poly(A,U) (random copolymer), poly(A-U) (alternating copolymer), poly(A) . poly(U) (double stranded), and poly(U) . poly(A) . poly(U) (triple stranded) were reacted with HMT at different temperatures and salt concentrations. The conformation of the polymers was monitored by UV absorption and circular dichroism. It was found that the rate of HMT photoaddition changed dramatically at structural transitions in the RNA. The alternating copolymer poly(A-U) was found to have the highest rate of addition. Low salt and temperature produced maximal incorporation.     

Poly(A) binding proteins located at the inner surface of resealed nuclear envelopes.

We have used a photoreactive cross-linking reagent, poly(A/8-N3-A) (a poly(A) of average molecular mass of 100 kDa in which 5-10% of the A residues are replaced by 8-N3-A), to label poly(A) binding proteins of rat liver nuclear envelopes. This reagent was prepared by polymerizing a mixture of ADP and 8-N3-ADP with polynucleotide phosphorylase. The purified poly(A) was labeled in the 5'-position with a 32P group. In nuclear envelopes prepared by a low salt DNase I procedure, the poly(A/8-N3-A) labeled a protein-nucleic acid complex of approximately 270 kDa, which on degradation with RNase U2 or NaOH at pH 10 yielded two polypeptides of approximately 50 and 30 kDa. These photoreaction products were markedly decreased when resealed nuclear envelopes or non-nuclear envelope proteins were irradiated in the presence of poly(A/8-N3-A). The affinity labeling was intensified when resealed vesicles were made leaky by freezing or ultrasonication, suggesting that the poly(A) binding proteins are accessible from the nucleoplasmic but not the cytoplasmic face of the envelope. Moreover binding was specific for poly(A). Alternative reagents, random poly(A/8-N3-A,C,G,U) of about 100 kDa and poly(dA) (molecular mass between 350 and 515 kDa), showed a very low affinity for poly(A) recognition proteins in the low salt DNase I-treated nuclear envelopes; the 270-kDa band was labeled only weakly. The binding site was not protected by poly(A,C,G,U), weakly by poly(dA), and distinctly by poly(A).