title: Selective glutaraldehyde-mediated coupling of proteins to the 3'-adenine terminus of polymerase chain reaction products.
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.
authors.affiliation: Operational Technologies Corporation, San Antonio, TX 78229, USA. john.bruno@otcorp.com
authors.forename: John G
authors.initials: JG
authors.lastname: Bruno
authors.forename: Randy
authors.initials: R
authors.lastname: Crowell
mesh_terms: D000225:Adenine / Q000737:chemistry*; D052157:Aptamers, Nucleotide / Q000138:chemical synthesis / Q000737:chemistry / Q000235:genetics; D001483:Base Sequence; D001709:Biotechnology; D003432:Cross-Linking Reagents; D004247:DNA / Q000737:chemistry* / Q000235:genetics*; D017931:DNA Primers / Q000138:chemical synthesis / Q000737:chemistry / Q000235:genetics; D024202:Electrophoretic Mobility Shift Assay; D005976:Glutaral; D020135:Peptide Nucleic Acids / Q000138:chemical synthesis / Q000737:chemistry / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*; D011506:Proteins / Q000737:chemistry*; D013050:Spectrometry, Fluorescence; D001613:beta 2-Microglobulin / Q000737:chemistry
chemical_list: D052157:Aptamers, Nucleotide; D003432:Cross-Linking Reagents; D017931:DNA Primers; D020135:Peptide Nucleic Acids; D011506:Proteins; D001613:beta 2-Microglobulin; D004247:DNA; D000225:Adenine; D005976:Glutaral