Selective glutaraldehyde-mediated coupling of proteins to the 3'-adenine terminus of polymerase chain reaction products.
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.
Bruno
John G
JG
Operational Technologies Corporation, San Antonio, TX 78229, USA. john.bruno@otcorp.com
Crowell
Randy
R
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
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