A versatile toolbox for variable DNA functionalization at high density.
To broaden the applicability of chemically modified DNAs in nano- and biotechnology, material science, sensor development, and molecular recognition, strategies are required for introducing a large variety of different modifications into the same nucleic acid sequence at once. Here, we investigate the scope and limits for obtaining functionalized dsDNA by primer extension and PCR, using a broad variety of chemically modified deoxynucleotide triphosphates (dNTPs), DNA polymerases, and templates. All natural nucleobases in each strand were substituted with up to four different base-modified analogues. We studied the sequence dependence of enzymatic amplification to yield high-density functionalized DNA (fDNA) from modified dNTPs, and of fDNA templates, and found that GC-rich sequences are amplified with decreased efficiency as compared to AT-rich ones. There is also a strong dependence on the polymerase used. While family A polymerases generally performed poorly on "demanding" templates containing consecutive stretches of a particular base, family B polymerases were better suited for this purpose, in particular Pwo and Vent (exo-) DNA polymerase. A systematic analysis of fDNAs modified at increasing densities by CD spectroscopy revealed that single modified bases do not alter the overall B-type DNA structure, regardless of their chemical nature. A density of three modified bases induces conformational changes in the double helix, reflected by an inversion of the CD spectra. Our study provides a basis for establishing a generally applicable toolbox of enzymes, templates, and monomers for generating high-density functionalized DNAs for a broad range of applications.
Jäger
Stefan
S
Kekuké-Institut für Organische Chemie und Biochemie, Universität Bonn, Gerhard-Domagk-Strasse 1, D-53121 Bonn, Germany.
Rasched
Goran
G
Kornreich-Leshem
Hagit
H
Engeser
Marianne
M
Thum
Oliver
O
Famulok
Michael
M
D001483:Base Sequence; D001709:Biotechnology; D002942:Circular Dichroism / Q000379:methods; D004247:DNA / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004259:DNA-Directed DNA Polymerase / Q000235:genetics / Q000378:metabolism*; D003854:Deoxyribonucleotides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D036103:Nanotechnology; D016133:Polymerase Chain Reaction / Q000379:methods; D011122:Polyphosphates / Q000737:chemistry; D013698:Templates, Genetic
D003854:Deoxyribonucleotides; D011122:Polyphosphates; D004247:DNA; D004259:DNA-Directed DNA Polymerase