title: Enzymatic synthesis of long double-stranded DNA labeled with haloderivatives of nucleobases in a precisely pre-determined sequence.
abstract: BACKGROUND
  Restriction endonucleases are widely applied in recombinant DNA technology. Among them, enzymes of class IIS, which cleave DNA beyond recognition sites, are especially useful. We use BsaI enzyme for the pinpoint introduction of halogen nucleobases into DNA. This has been done for the purpose of anticancer radio- and phototherapy that is our long-term objective.
  
  
  RESULTS
  An enzymatic method for synthesizing long double-stranded DNA labeled with the halogen derivatives of nucleobases (Hal-NBs) with 1-bp accuracy has been put forward and successfully tested on three different DNA fragments containing the 5-bromouracil (5-BrU) residue. The protocol assumes enzymatic cleavage of two Polymerase-Chain-Reaction (PCR) fragments containing two recognition sequences for the same or different class IIS restriction endonucleases, where each PCR fragment has a partially complementary cleavage site. These sites are introduced using synthetic DNA primers or are naturally present in the sequence used. The cleavage sites are not compatible, and therefore not susceptible to ligation until they are partially filled with a Hal-NB or original nucleobase, resulting in complementary cohesive end formation. Ligation of these fragments ultimately leads to the required Hal-NB-labeled DNA duplex. With this approach, a synthetic, extremely long DNA fragment can be obtained by means of a multiple assembly reaction (n × maximum PCR product length: n × app. 50 kb).
  
  
  CONCLUSIONS
  The long, precisely labeled DNA duplexes obtained behave in very much the same manner as natural DNA and are beyond the range of chemical synthesis. Moreover, the conditions of synthesis closely resemble the natural ones, and all the artifacts accompanying the chemical synthesis of DNA are thus eliminated. The approach proposed seems to be completely general and could be used to label DNA at multiple pre-determined sites and with halogen derivatives of any nucleobase. Access to DNAs labeled with Hal-NBs at specific position is an indispensable condition for the understanding and optimization of DNA photo- and radio-degradation, which are prerequisites for clinical trials of Hal-NBs in anticancer therapy.
authors.affiliation: Department of Chemistry University of Gdańsk, Sobieskiego 18, 80-952 Gdańsk, Poland.
authors.forename: Ireneusz
authors.initials: I
authors.lastname: Sobolewski
authors.forename: Katarzyna
authors.initials: K
authors.lastname: Polska
authors.forename: Agnieszka
authors.initials: A
authors.lastname: Zylicz-Stachula
authors.forename: Joanna
authors.initials: J
authors.lastname: Jeżewska-Frąckowiak
authors.forename: Janusz
authors.initials: J
authors.lastname: Rak
authors.forename: Piotr
authors.initials: P
authors.lastname: Skowron
mesh_terms: D020029:Base Pairing; D001483:Base Sequence; D001973:Bromodeoxyuridine / Q000378:metabolism; D004247:DNA / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics*; D004262:DNA Restriction Enzymes / Q000378:metabolism*; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D006219:Halogens / Q000737:chemistry*; D009693:Nucleic Acid Hybridization
chemical_list: D006219:Halogens; D004247:DNA; D004259:DNA-Directed DNA Polymerase; D004262:DNA Restriction Enzymes; D001973:Bromodeoxyuridine