Abstract
Attachment of proteins to the 3' end of DNA increases stability of the DNA in serum and retards clearance of DNA by major organs, thereby enhancing in vivo half-life and therapeutic potential of DNA. Unfortunately, the length of DNA molecules that can be produced with 3 ' modifications by solid-phase synthesis for protein attachment is limited to 45-60 nucleotides due to uncertainties about sequence fidelity for longer oligonucleotides. Here we describe selective covalent coupling of proteins or other molecules to the 3'-adenine overhang of unlabeled and fluorophore-labeled double-stranded polymerase chain reaction products putatively at the N6 position of adenine using 2.5% glutaraldehyde at pH 6.0 and 4 degrees C for at least 16 h. Gel mobility shift analyses and fluorescence analyses of the shifted bands supported conjugate formation between double-stranded polymerase chain reaction products and beta2-microglobulin. In addition, blunt-ended DNA ladder fragments treated with glutaraldehyde at 4 degrees C showed no evidence of DNA-DNA or DNA-protein conjugate formation. With the present cold glutaraldehyde technique, longer DNA-3'-protein conjugates might be easily mass-produced. The protein portion of a DNA-3'-protein conjugate could possess functionality as well, such as receptor binding for cell entry, cytotoxicity, or opsonization.