One of the goals in synthetic biology is to design novel base pairs that ultimately could be used to extend the genetic code within a living organism. In order to achieve this goal, the novel base pairs would ideally be accommodated within normal helical forms of DNA such as A- and B-form DNA. The focus of the present study is the structural characterization of 16 base pair oligonucleotides including 2-6 consecutive Z-P base pairs. This particular novel base pair includes the pyrimidine-like base Z, 6-amino-5- nitro-3(1’-β-D-2’-deoxyribofuranosyl)-2 (1H)-pyridone, and the purine-like base P, 2-amino-8-(1’-β-D-2’-deoxyribofuranosyl(-imidazo[1,2,-<i>a</i>]-1,3,5-triazin-4(8H)-one and has an altered hydrogen-bonding pattern. A single novel base pair can likely be accommodated within a DNA duplex. Thus, we were most interested in determining the structural consequences of including 2 to 6 consecutive base pairs. Our initial strategy for obtaining crystals was to use our host-guest system in which the N-terminal fragment of Moloney murine leukemia virus reverse transcriptase serves as a host for a 16 base pair duplex oligonucleotide. In this system, B-form DNA will naturally be selected through the crystallization process due to lattice constraints. Of those screened, the oligonucleotide containing 2 consecutive ZP pairs crystallized in the host-guest system, and its structure was determined by molecular replacement and refined to 1.7 Å. The DNA in this structure is B-form. Further screening of the remaining oligonucleotides produced crystals of the oligonucleotide containing 6 consecutive ZP base pairs (3/6 ZP) in a novel lattice. This structure was determined by Br SAD phasing methods and refined to 1.98 Å resolution. The 3/6 ZP structure is A-form DNA. The 3/6 ZP oligonucleotide was further characterized by CD and found to exhibit features similar to those of an oligonucleotide in which the ZP pairs were replaced with GC pairs but also had some unique features. These studies will form the basis for understanding the properties of ZP base pairs within duplex DNA and potentially the basis for recognition by proteins.

Structural characterization of non-natural ZP base pairs in duplex DNA

249th National Meeting (2015)

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