Crosslinking nucleic acid-protein

Russmann C, Stollhof J, Weiss C, Beigang R, Beato M (1998) Two wavelength femtosecond laser induced DNA-protein crosslinking. Nucleic Acids Research 26 3967-3970. 

Gels have crosslinked three-dimensional (3D) structures that are actively used in the biomaterial field for their diverse encapsulation ability, including for hydrophobic/hydrophilic drugs, nucleic acids, proteins and metal/inorganic particles [47, 48]. Shimoda and... 

Numerous organisms, both marine and terrestrial, produce protein toxins. These are typically relatively small, and rich in disulfide crosslinks. Since they are often difficult to crystallize, relatively few structures from this class of proteins are known. In the past five years two dimensional NMR methods have developed to the point where they can be used to determine the solution structures of small proteins and nucleic acids. We have analyzed the structures of toxins II and III of RadiarUhus paumotensis using this approach. We find that the dominant structure is )9-sheet, with the strands connected by loops of irregular structure. Most of the residues which have been determined to be important for toxicity are contained in one of the loops. The general methods used for structure analysis will be described, and the structures of the toxins RpII and RpIII will be discussed and compared with homologous toxins from other anemone species. 

Chemical attachment of a detectable component to an oligonucleotide forms the basis for constructing a sensitive hybridization reagent. Unfortunately, the methods developed to crosslink or label other biological molecules such as proteins do not always apply to nucleic acids. The major reactive sites on proteins involve primary amines, sulfhydryls, carboxylates, or phenolates— groups that are relatively easy to derivatize. RNA and DNA contain none of these functionalities. 

The process was later refined for photo-polymerization of both nucleic acids and proteins. The issues of slowed diffusion and exclusion of targets because of limited porosity were addressed by substituting diallyltartardiamide as the crosslinker in place of bis-acrylamide (Guschin et al., 1997). Finally,... 

Sephadex, which can also be obtained in a variety of ion-exchange forms (see Table 15) consists of beads of a crosslinked dextran gel which swells in water and aqueous salt solutions. The smaller the bead size the higher the resolution that is possible but the slower the flow rate. Typical applications of Sephadex gels are the fractionation of mixtures of polypeptides, proteins, nucleic acids, polysaccharides and for desalting solutions. 

Photochemically reactive molecules have often been used as labels for specific sites in proteins and nucleic acids. Psoralen derivatives serve as relatively nonspecific photochemically activated crosslinking agents for DNA and double-stranded RNA.195 Aryl azides are converted by light to aryl nitrenes, which react in a variety of ways including insertion into C-H bonds (Eq. 23-27).200 201 In some cases UV irradiation can be used to join natural substrates to enzymes or hormones to receptors. For example, progesterone, testosterone, and other steroids have been used for direct photoaffinity labeling of their receptors.202 Synthetic benzophenones have also been used widely as photoactivated probes.203... 

Formaldehyde is a toxic substance that reacts spontaneously with amino groups of proteins and nucleic acids, hydroxymethylating them and forming methylene-bridge crosslinks between them. Free formaldehyde therefore wreaks havoc in living cells and could not serve as a useful hydroxymethylating agent. In the form of A5,A10-methylenetetrahydrofolate, however, its chemical reactivity is attenuated but retained in a potentially available form where needed for specific enzymatic action. Formate, how-... 

On the other hand, available evidence supports the occurrence of the breakage of protein crosslinks, which allows the antibody access to the antigen. Conventional heat, dry or steam, breaks down reversible protein-protein, protein-nucleic acid, and protein-carbohydrate crosslinks introduced by formaldehyde and thereby unmasks the epitopes, as well as allowing the antibodies access to the epitopes. It is well known that most, if not all, crosslinks formed during formaldehyde fixation are destroyed upon heating even at 37°C... 

The role of high temperatures in the breakdown of protein crosslinks introduced by aldehydes has been discussed earlier in this book. The aforementioned combined technique is especially effective in increasing the in situ hybridization signal in archival tissues, the fixation history of which may or may not be known. This protocol also detects low levels of nucleic acids in the tissue, which may not be detectable with heating or enzyme digestion alone. 

The demonstration of stepwise adduct formation by psoralens was an important advance. At 390 nm monoadduct formation occurs a crosslink is completed by irradiation at 360 nm (Chatterjee and Cantor, 1978). DNA-psoralen monoadducts have been used to covalently label complementary sites on large RNA molecules. The sites may then be mapped by electron microscopy of the denatured nucleic acid complexes (Wollenzien and Cantor, 1982). Psoralen derivatives for attachment to proteins have been made (Cantor, 1980) and this will allow the exploration of protein binding sites on nucleic acids by photochemical crosslinking. 

For maximizing protein yield and minimizing contaminant nucleic acids, alkali extraction at elevated temperatures is a feasible procedure (63,73). However denaturation of proteins during extraction is a serious problem because it significantly destroys functional properties and limits the food uses of the extracted protein (2, 74). In addition to denaturation, exposure of protein to alkaline treatments may also cause other undesirable effects, i.e. racemization of amino acids, g-elimination and crosslinking or certain amino acids and formation of potentially antinutritive compounds (11,23,75). 

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