CyDNA: synthesis and replication of highly Cy-dye substituted DNA by an evolved polymerase.
DNA not only transmits genetic information but can also serve as a versatile supramolecular scaffold. Here we describe a strategy for the synthesis and replication of DNA displaying hundreds of substituents using directed evolution of polymerase function by short-patch compartmentalized self-replication (spCSR) and the widely used fluorescent dye labeled deoxinucleotide triphosphates Cy3-dCTP and Cy5-dCTP as substrates. In just two rounds of spCSR selection, we have isolated a polymerase that allows the PCR amplification of double stranded DNA fragments up to 1kb, in which all dC bases are substituted by its fluorescent dye-labeled equivalent Cy3- or Cy5-dC. The resulting "CyDNA" displays hundreds of aromatic heterocycles on the outside of the DNA helix and is brightly colored and highly fluorescent. CyDNA also exhibits significantly altered physicochemical properties compared to standard B-form DNA, including loss of silica and intercalating dye binding, resistance to cleavage by some endonucleases, an up to 40% increased apparent diameter as judged by atomic force microscopy and organic phase partitioning during phenol extraction. CyDNA also displays very bright fluorescence enabling significant signal gains in microarray and microfluidic applications. CyDNA represents a step toward a long-term goal of the encoded synthesis of DNA-based polymers of programmable and evolvable sequence and properties.
MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 0QH, United Kingdom.
Nicola
N
Ramsay
Ann-Sofie
AS
Jemth
Anthony
A
Brown
Neal
N
Crampton
Paul
P
Dear
Philipp
P
Holliger
D004396:Coloring Agents / Q000737:chemistry*; D004247:DNA / Q000096:biosynthesis* / Q000737:chemistry*; D004261:DNA Replication; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D008958:Models, Molecular; D009711:Nucleotides / Q000737:chemistry / Q000378:metabolism
D004396:Coloring Agents; D009711:Nucleotides; D004247:DNA; D004259:DNA-Directed DNA Polymerase