Oligonucleotides crosslinking methods

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 unique properties of oligonucleotides create crosslinking options that are far different from any other biological molecule. Nucleic acids are the only major class of macromolecule that can be specifically duplicated in vitro by enzymatic means. The addition of modified nucleoside triphosphates to an existing DNA strand by the action of polymerases or transferases allows addition of spacer arms or detection components at random or discrete sites along the chain. Alternatively, chemical methods that modify nucleotides at selected functional groups can be used to produce spacer arm derivatives or activated intermediates for subsequent coupling to other molecules. 

Generally, DNA complexation to such particles can be performed either by direct crosslinking of PEI/DNA polyplexes with chosen amine-reactive crosslinkers or by complexing the oligonucleotides or DNA by pre-crosslinked PEI particles. It is important to mention that the latter method often yields low molecular weight... 

Since the introduction of sohd-phase peptide synthesis (SPPS) on lightly crosslinked polystyrene (PSty) by Merrifield in 1962,this technique has been optimized in all its aspects and has subsequently found widespread application in many other fields of organic chemistry, e.g. for the synthesis of oligonucleotides, for catalysis, for the immobilization of enzymes in biotechnology, and for the preparation of polymeric reagents. A new but very rapidly growing field of application of this revolutionary method is the use of solid-phase techniques in combinatorial chemistry for the synthesis of hbraries of compounds whose constituents differ widely in their chemical nature. ... 

Crosslinking of DNA and proteins is a versatile method to study protein-DNA interactions. Among others, phosphoramidites of brominated or iodinated 2 -deoxycytidines [325], 2 -desoxuridines [326,327] and recently also of 2 -deoxyadenosines 132 have been used [328]. Nucleophilic replacement of the bromine by an amino group during deprotection does not occur when reaction conditions of concentrated ammonia solution for 24 h at room temperature are used after oligonucleotide synthesis. 

Seleno-thymidine was incorporated onto the 5 -end of an oligonucleotide, where, due to its high nucleophilicity, it was used as a method for labelling DNA, exemplified by the use of fluorescein. The C5 -oxidised thymidine lesion (19) was synthesised and incorporated into DNA to study its biochemical properties. It was found that the lesion was excised by DNA polymerase (3 in the presence of flap endonuclease 1. A C5 -boronic acid derivative has been described that will form a crosslink via a 2, 3 -borate ester with a terminal opposing ribonucleotide. ... 

DNA-modified gold nanoparticles have been used as drug delivery vectors for doxorubicin as well as for oligonucleotides. " An electrochemical method is reported for the detection of DNA methylation in an assay for methyltransferase activity," and various assays are described for nuclease assays." " Multimerised siRNA crosslinked by gold... 

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