UV Light-induced Cross-linking Research Articles

Single-Component Hydrophilic Terpolymer Thin Film Systems for Imparting Surface Chemical Versatility on Various Substrates.

We demonstrate a single-component hydrophilic photocrosslinkable copolymer system that incorporates all critical functionalities into one chain. This design allows for the creation of uniform functional organic coatings on a variety of substrates. The copolymers were composed of a poly(ethylene oxide)-containing monomer, a monomer that can release a primary amine upon UV light, and a monomer with reactive epoxide or cyclic dithiocarbonate with a primary amine. These copolymers are easily incorporated into the solution-casting process using polar solvents. Furthermore, the resulting coating can be readily stabilized through UV light-induced crosslinking, providing an advantage for controlling the surface properties of various substrates. The photocrosslinking capability further enables us to photolithographically define stable polymer domains in a desirable region. The resulting copolymer coatings were chemically versatile in immobilizing complex molecules by (i) post-crosslinking functionalization with the reactive groups on the surface and (ii) the formation of a composite coating by mixing varying amounts of a protein of interest, i.e., fish skin gelatin, which can form a uniform dual crosslinked network. The number of functionalization sites in a thin film could be controlled by tuning the composition of the copolymers. In photocrosslinking and subsequent functionalizations, we assessed the reactivity of the epoxide and cyclic dithiocarbonate with the generated primary amine. Moreover, the orthogonality of the possible reactions of the presented reactive functionalities in the crosslinked thin films with complex molecules is assessed. The resulting copolymer coatings were further utilized to define a hydrophobic surface or an active surface for the adhesion of biological objects.

Novel Cross-Linked Gel Polymer Electrolyte (GPE) with Long-Term Cycle Life for Li-Metal Batteries

Lithium metal batteries (LMBs) are considered among the most promising energy storage technologies due to their very high energy density and lightweight. While liquid electrolytes (LEs) offer high ionic conductivities (10−3−10−2 S cm−1) and low interfacial resistance with electrodes, yet the risks of leakage and combustion of organic solvents, lithium (Li) dendrite formation, thermal runaway, and environmental concerns regarding using organic solvents hinder their full potential applications. Li dendrites are mainly induced by nonuniform distribution of charge or ions and formation of a fragile, unstable solid electrolyte interphase (SEI) layer on Li metal anode during electrochemical Li deposition. The dendritic morphology of electrochemically deposited Li results in inefficient lithiation/delithiation and the formation of inactive or dead Li and constant degradation of organic solvents during Li plating/stripping, leading to capacity decade and decline in cycle life. Furthermore, lithium dendrites can pose a safety issue by penetrating through a porous or mechanically soft separator and making a short-circuit. High voltage instability of conventional electrolytes is another issue restricting application of high potential cathodes to enhance the energy density of LMBs. Gel polymer electrolytes (GPEs) are considered a promising alternative for LEs because of their high ionic conductivities (10−3−10−2 S cm−1), prevention of liquid leakage, low flammability, chemical and electrochemical stability, high flexibility for portable and wearable electronics, suppression of dendrite formation, and ample chemistry and design [1-4].In this work, we have developed a novel, highly efficient PEO-based GPE by a simple, facile chemical method and using UV light-induced cross-linking. Cross-linking is essential to retain the structure of the polymer electrolyte and it is also shown to suppress Li dendrite growth by offering a high stiffness and rigidity (high shear modulus) toward Li dendrites. The designed GPE owns a very high ionic conductivity similar to commercial LEs, electrochemical stability up to 5 V, long-term cycle life and stability. While Li|Li symmetric cells made with 1 M LiPF6 in EC/DEC and 1 M LiTFSI in DOL/DME LEs showed an increase in overpotential during cycling (mainly due to the formation of dead Li and evolution of SEI) followed by a short-circuit (probably due to penetration of Li dendrites through Celgard separator), Li|Li cells made with GPE exhibit a long-term stability with a low voltage polarization of less than 0.1 V at a high current density of 0.5 mAh cm−2 for 500 cycles. Furthermore, Li|LFP full cells made with GPE display a long-term cycle life with discharge capacity of ~120 mAh g−1 at 0.5 C in ambient temperature with around 80% capacity retention after 500 cycles.In situ light microscopy which is a simple, versatile technique to study the nucleation and growth of Li particles during electrochemical platting/stripping was also used to further illustrate uniform deposition of Li by the developed GPEs. Hence, we have also performed light microscopy analysis to investigate Li deposition in Li|Li cells made of LEs and GPEs during electrochemical deposition process. It was observed that unlike LEs made of 1 M LiPF6 in EC/DEC and 1 M LiTFSI in DOL/DME which results in non-uniform, dendritic growth of Li, a uniform and non-dendritic deposition of Li was observed in case of GPE, which can be due to uniform regulation of Li through complexation with oxygen atoms of the PEO-based GPE.

Genomic DNA Regions Whose Complementary Strands Are Prone to UV Light-Induced Crosslinking

We prepared an extensive set of DNA restriction fragments, irradiated them with UV light, and detected crosslinked complementary strands by electrophoresis in denaturing agarose gels. These experimental data were quantified by densitometry to determine tetranucleotide contributions to crosslinking. The tetranucleotide contributions were used to predict genomic maps of the crosslinking probability that permitted us to identify two strongly crosslinking genomic regions having 295 and 389 base pairs in length. The two sequences shared the (ATTTTATA).(TATAAAAT) octamer, which is a candidate for the hotspot of UV light-induced crosslinking between the complementary strands of DNA.

UV Light-induced Cross-linking of Nucleosides, Nucleotides and a Dinucleotide to the Carboxy-terminal Heptad Repeat Peptide of RNA Polymerase II as Studied by Mass Spectrometry

Abstract— We report here the results of a study to assess the usefulness of mass spectrometry as a method for rapidly locating cross-linking sites in peptides modified by UV irradiation in the presence of nucleic acid components. For this study, we selected two nucleosides (thymidine and 5-bromo-2′-deoxyuridine), two nucleotides (thymi-dine-5′-monophosphate and 5-bromo-2′-deoxyuridine-5-monophosphate) and a dinucleotide (thymidylyl-[3′→5′]-2′-deoxyadenosine). The peptide picked was SPSYSPT (l-seryl-l-prolyl-l-seryl-l-tyrosyl-l-seryl-l-prolyl-l-threonine), the heptad repeat unit found in the largest subunit of the RNA polymerase II multiprotein complex. Modified peptides were isolated by reversed-phase HPLC. Molecular mass measurements confirmed that covalent adducts had been formed. High-energy tandem collision-induced dissociation mass spectrometry pinpointed the location of cross-linking in each modified peptide as being at the tyrosine residue. These results indicate that mass spectrometry is a potentially applicable technique for location of cross-linking sites in peptides, modified by attachment of nucleosides, nucleotides and dinucleotides. Such modified peptides would be among the products expected after application of standard proteolytic and nucleolytic digestion protocols to digestion of cross-linked DNA-protein complexes.

UV light-induced crosslinking of the complementary strands of plasmid pUC19 DNA restriction fragments.

Restriction fragments of pUC19 DNA were irradiated by various doses of UV light and analyzed by denaturing (alkaline) agarose gel electrophoresis. The irradiation generated retarded species whose mobility indicated two crosslinked DNA strands. Quantitative analysis of the experimental data provided an empirical equation relating the fraction of crosslinked DNA molecules to their length and to the dose of their irradiation by UV light. This equation can be used to predict the crosslinking behavior of pUC19-like DNA molecules whose primary structures do not much differ from a random nucleotide sequence. The amount of interstrand crosslinks increased with the (A+T) content of the pUC19 DNA fragments but the dependence was not clear-cut to indicate that oligonucleotide composition of DNA played a significant role as well.

UV light-induced crosslinking of short DNA duplex strands: nucleotide sequence preferences and a prominent role of the duplex ends.

By various doses of UV light, we irradiated 32 short DNA duplexes having between 12 and 40 nucleotide residues in length, and monitored the induced crosslinks between the complementary DNA strands by denaturing polyacrylamide gel electrophoresis. The experiments revealed that the crosslinking was strongest with the alternating sequence of T and A and weaker with the alternating sequence of T and G (C and A in the complementary strand). On the other hand, GC blocks of any sequence provided undetectable amounts of interstrand crosslinks even at the highest doses of UV irradiation. The amount of crosslinked strands logarithmically increased with the UV dose but it did not depend on the oligonucleotide concentration, ionic strength, divalent magnesium or manganese cations and pH at least within the examined regions of the experimental conditions, unless the oligonucleotide denatured or isomerized into a unimolecular foldback. The extent of crosslinking also did not depend on the (dT-dA)n duplex length to indicate that the crosslink was predominantly localized at a specific duplex locus. Experiments with (dT-dA)8 "mutants" in which AT pairs were systematically replaced by GC pairs at various molecule positions, revealed that the crosslinking predominantly occurred at the oligo(dT-dA) duplex ends. The crosslinking is a direct method to detect duplexes of DNA, which is here, for example, demonstrated with the heteroduplex of (dT-dA)12 and (dT-dA)16.

Combined application of in vivo UV-crosslinking and in vitro label transfer in the examination of AU-rich sequence binding protein - RNA interactions.

A combination of in vivo UV light-induced crosslinking of nucleic acids to proteins and in vitro label transfer assay was applied to investigate specific interactions between AU-rich sequences (ARE) in the 3′ UTR of lymphokine mRNAs and cytoplasmic AU-rich sequence element binding proteins (AUBP) in normal human lymphoblasts and MLA 144 gibbon lymphoid tumor cells. We demonstrate that a pool of cytoplasmic AUBP can be effectively crosslinked to RNA in vivo, suggesting a close association of these proteins with ARE sequences in the cytoplasm. We also show that the UV-crosslinked AUBP pool is markedly reduced in malignantly transformed MLA 144 cells compared with normal lymphoblasts, indicating weaker interaction between lymphokine ARE and AUBP in these tumor cells. Similar differences in AUBP-RNA associations were found between the membrane-bound polysomal subfractions of the two cell types where most of the AUBP activity was localized. We suggest that the decreased AUBP-mRNA association in MLA 144 cells might reflect a process concerned with disturbances of mRNA metabolism in the neoplastic phenotype.

Identification of an RNA binding region within the N-terminal third of the influenza A virus nucleoprotein

The influenza A virus nucleoprotein (NP) has been examined with regard to its RNA-binding characteristics. NP, purified from virions and devoid of RNA, bound synthetic RNAs in vitro and interacted with the ribonucleotide homopolymers poly(A), poly(G), poly(U), and poly(C) in a salt-dependent manner, showing higher binding affinity for polypyrimidine homopolymers. To map the NP regions involved in RNA binding, a series of deleted forms of the NP were prepared, and these truncated polypeptides were tested for their ability to bind poly(U) and poly(C) homopolymers linked to agarose beads. Proteins containing deletions at the N terminus of the NP molecule showed reduced RNA-binding activity, indicating that this part of the protein was required to bind RNA. To identify the NP region or regions which directly interact with RNA, proteins having the maltose-binding protein fused with various NP fragments were obtained and tested for binding to radioactively labeled RNAs in three different assays: (i) nitrocellulose filter binding assays, (ii) gel shift assays, and (iii) UV light-induced cross-linking experiments. A maltose-binding protein fusion containing the N-terminal 180 amino acids of NP behaved as an RNA-binding protein in the three assays, demonstrating that the N terminus of NP can directly interact with RNA. This NP region could be further subdivided into two smaller regions (amino acids 1 to 77 and 79 to 180) that also retained RNA-binding activity.

UV Light-Induced Crosslinking of the Strands of Poly(dA-dT) and Related Alternating Purine-Pyrimidine DNAs

Alkaline agarose gel electrophoresis was used to detect UV-induced crosslinking of the strands of poly(dA-dT) and related alternating purine-pyrimidine DNAs in solutions stabilizing various polynucleotide conformations. Strands of the B-form and A-form of poly(dA-dT) were not crosslinked but a UV dose-dependent retarded species appeared in the denaturing gels in parallel with the polynucleotide isomerization into the unusual X-form. Most other polynucleotides adopting the X-form were crosslinked as well. The exceptions include the X-forms of poly(dA-butyl5dU) and poly(dA-pentyl5dU) whose strands do not crosslink because the long exocyclic substituents attached to uracil make the photodimerization impossible. Strands of poly(amino2dA-dT) and poly(dA, amino2dA-dT), the latter polynucleotide containing roughly equal amounts of amino2adenine and adenine, also do not crosslink upon UV irradiation because they isomerize into an A-like conformation which is different from the X-form of poly (dA-dT). In contrast, strands of the mixed copolymers of poly(dA, amino2dA-dT) containing low amino2adenine contents are crosslinked upon UV irradiation, in accordance with the observation that they isomerize into the X-form.

NAB2: a yeast nuclear polyadenylated RNA-binding protein essential for cell viability.

A variety of nuclear ribonucleoproteins are believed to associate directly with nascent RNA polymerase II transcripts and remain associated during subsequent nuclear RNA processing reactions, including pre-mRNA polyadenylation and splicing as well as nucleocytoplasmic mRNA transport. To investigate the functions of these proteins by using a combined biochemical and genetic approach, we have isolated nuclear polyadenylated RNA-binding (NAB) proteins from Saccharomyces cerevisiae. Living yeast cells were irradiated with UV light to covalently cross-link proteins intimately associated with RNA in vivo. Polyadenylated RNAs were then selectively purified, and the covalent RNA-protein complexes were used to elicit antibodies in mice. Both monoclonal and polyclonal antibodies which detect a variety of NAB proteins were prepared. Here we characterize one of these proteins, NAB2. NAB2 is one of the major proteins associated with nuclear polyadenylated RNA in vivo, as detected by UV light-induced cross-linking. Cellular immunofluorescence, using both monoclonal and polyclonal antibodies, demonstrates that the NAB2 protein is localized within the nucleus. The deduced primary structure of NAB2 indicates that it is composed of at least two distinct types of RNA-binding motifs: (i) an RGG box recently described in a variety of heterogeneous nuclear RNA-, pre-rRNA-, mRNA-, and small nucleolar RNA-binding proteins and (ii) CCCH motif repeats related to the zinc-binding motifs of the largest subunit of RNA polymerases I, II, and III. In vitro RNA homopolymer/single-stranded DNA binding studies indicate that although both the RGG box and CCCH motifs bind poly(G), poly(U), and single-stranded DNA, the CCCH motifs also bind to poly(A). NAB2 is located on chromosome VII within a cluster of ribonucleoprotein genes, and its expression is essential for cell growth.

Photochemical Cross-Linking of Cap Binding Proteins to Eucaryotic mRNAs: Effect of mRNA 5′ Secondary Structure

We used UV light-induced cross-linking to study the interactions of cap binding proteins with the 5' cap structure of eucaryotic mRNAs. Thymidine kinase gene (herpes simplex virus type 1) transcripts prepared in vitro using the SP6 RNA polymerase transcription system were capped and methylated posttranscriptionally with [alpha-32P]GTP and S-adenosyl-L-methionine to yield cap-labeled transcripts. Irradiation of capped transcripts with crude rabbit reticulocyte initiation factors in the presence of ATP-Mg2+ resulted in the cap-specific cross-linking of two polypeptides with molecular masses of 24 and 80 kilodaltons (kDa). The cross-linking characteristics of these polypeptides resemble those of the cap-binding proteins previously detected by a chemical cross-linking assay (N. Sonenberg, D. Guertin, D. Cleveland, and H. Trachsel, Cell 27:563-572, 1981). However, the relative efficiency of the cross-linking of these two polypeptides to the cap structure was different from that in previous studies, and there was no detectable cross-linking of the previously described 50-kDa polypeptide. In addition, we present data indicating that the insertion of secondary structure into the 5' noncoding region of tk mRNA, 6 nucleotides from the cap structure, decreases the cap-specific cross-linking of the 80-kDa but not the 24-kDa polypeptide. In contrast, the insertion of secondary structure 37 nucleotides from the cap structure had no significant effect on the cross-linking of either the 24- or the 80-kDa cap-specific polypeptide. These results demonstrate that the position of mRNA 5'-proximal secondary structure relative to the cap structure can influence the cap-specific interaction between the mRNA and a translation initiation factor.

Photochemical cross-linking of cap binding proteins to eucaryotic mRNAs: effect of mRNA 5' secondary structure.

We used UV light-induced cross-linking to study the interactions of cap binding proteins with the 5' cap structure of eucaryotic mRNAs. Thymidine kinase gene (herpes simplex virus type 1) transcripts prepared in vitro using the SP6 RNA polymerase transcription system were capped and methylated posttranscriptionally with [alpha-32P]GTP and S-adenosyl-L-methionine to yield cap-labeled transcripts. Irradiation of capped transcripts with crude rabbit reticulocyte initiation factors in the presence of ATP-Mg2+ resulted in the cap-specific cross-linking of two polypeptides with molecular masses of 24 and 80 kilodaltons (kDa). The cross-linking characteristics of these polypeptides resemble those of the cap-binding proteins previously detected by a chemical cross-linking assay (N. Sonenberg, D. Guertin, D. Cleveland, and H. Trachsel, Cell 27:563-572, 1981). However, the relative efficiency of the cross-linking of these two polypeptides to the cap structure was different from that in previous studies, and there was no detectable cross-linking of the previously described 50-kDa polypeptide. In addition, we present data indicating that the insertion of secondary structure into the 5' noncoding region of tk mRNA, 6 nucleotides from the cap structure, decreases the cap-specific cross-linking of the 80-kDa but not the 24-kDa polypeptide. In contrast, the insertion of secondary structure 37 nucleotides from the cap structure had no significant effect on the cross-linking of either the 24- or the 80-kDa cap-specific polypeptide. These results demonstrate that the position of mRNA 5'-proximal secondary structure relative to the cap structure can influence the cap-specific interaction between the mRNA and a translation initiation factor.

UV light-induced cross-linking of strychnine to the glycine receptor of rat spinal cord membranes

Summary The glycine receptor antagonist,[3H]strychnine, was bound irreversibly to rat spinal cord membranes upon UV illumination. The incorporation of [3H]strychnine into these membranes could be inhibited by glycine. Polyacrylamide gel electrophoresis of the labelled membranes in the presence of sodium dodecyl sulphate showed that[3H]strychnine was linked to a single polypeptide of an apparent molecular weight of 48,000. This polypeptide may be associated with the chloride ionophore of the glycine receptor.