Ferrocene conjugates of dUTP for enzymatic redox labelling of DNA

Two ferrocene-labelled analogues of dTTP, 5-(3-ferrocenecarboxamidopropenyl-1) 2'-deoxyuridine 5'-triphosphate (Fc1-dUTP) and 5-(3-ferroceneacetamidopropenyl-1) 2'-deoxyuridine 5'-triphosphate (Fc2-dUTP) have been produced to demonstrate the Incorporation of redox labels Into DNA by polymerases. Cyclic voltammetry Indicates that the ferrocenyl moieties display reversible redox behaviour in aqueous buffer with E-1/2 values of 398 (Fc1-dUTP) and 260 mV (Fc2-dUTP) versus Ag/AgCl. Primer extension by the proofreading enzymes Klenow fragment and T4 DNA polymerase shows that Fc1-dUTP Is efficiently incorporated Into DNA during synthesis, Including incorporation of two successive modified nucleotides. Production of a 998 bp amplicon by Tth DNA polymerase demonstrates that Fc1-dUTP Is also a satisfactory substrate for PCR. Despite Its structural similarity, Fc2-dUTP acts predominantly as a terminator with the polymerases employed here. UV melting analysis of a 37mer duplex containing five Fc1-dU residues reveals that the labelled nucleotide Introduces only a modest helix destabilisation, with T-m = 71 versus 75degreesC for the corresponding natural construct. Modified DNA is detected at femtomole levels using a HPLC system with a coulometric detector. The availability of simple and effective enzymatic labelling strategies should promote the further development of electrochemical detection In nucleic acid analysis.