 |
|
Title
|
|
CyDNA: synthesis and replication of highly Cy-dye substituted DNA by an evolved polymerase.
|
|
|
Authors
|
|
N.Ramsay,
A.S.Jemth,
A.Brown,
N.Crampton,
P.Dear,
P.Holliger.
|
|
|
Ref.
|
|
J Am Chem Soc, 2010,
132,
5096-5104.
|
|
|
PubMed id
|
|
|
|
|
|
Abstract
|
|
|
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.
|
|
|
|