Structural Basis for a Six Nucleotide Genetic Alphabet

Abstract

The extent to which synthetic biology can be used to expand genetic information systems compatible with natural enzymes and cells will depend on the extent to which multiple and contiguous non-natural nucleobase pairs fit within the standard double helical conformations of DNA. Toward this goal, two non-standard nucleobases (Z, 6-amino-5-nitro-2(1H)-pyridone and P, 2-amino-imidazo[1,2-a]-1,3,5-triazin-4(8H)one) were designed to form a Z:P pair with a standard “edge on” Watson-Crick geometry, but with rearranged hydrogen bond donor and acceptor groups. Here, we present the crystal structures of two self-complementary 16-mer oligonucleotides containing Z:P pairs. The first contained two consecutive Z:P nucleobase pairs and was found to crystallize within a host-guest complex in B-form. The second contained six consecutive Z:P pairs; it was found to crystallize as an A-form DNA duplex, although it can adopt B-form in solution as inferred from circular dichroism spectra. Although Z:P pairs have some structural properties that are similar to those of G:C pairs, unique features include stacking of the nitro group on Z with the adjacent heterocyclic nucleobase ring in A-DNA. In both B-and A-DNA, major groove widths associated with the Z:P pairs are approximately 1 Å wider than those of comparable G:C pairs potentially due to the presence of the nitro group in Z. Thus, our structural studies suggest that multiple and consecutive Z:P pairs are readily accommodated in DNA duplex structures recognized by natural polymerases, and therefore the GACTZP synthetic genetic system has the requisite properties to expand sequence space.

Structure and Biophysics for a Six Letter DNA Alphabet that Includes Imidazo[1,2-a]-1,3,5-triazine-2(8H)-4(3H)-dione (X) and 2,4-Diaminopyrimidine (K)

“…On the other hand, P : Z hydrogen bonding distances at position 6 for N2-O2, N1-N3, and O6-N4 are 2.57, 2.79, and 2.94 Å, respectively, and the values for position 7 P : Z are 2.63, 2.80, and 2.90 Å, respectively 11 . This finding is consistent with stronger theoretical hydrogen bonding reported for P : Z 41 than G:C or A:T. P : Z pairs have also been shown to be more stable than possible mispairs experimentally 42 .…”

Snapshots of an evolved DNA polymerase pre- and post-incorporation of an unnatural nucleotide

“…All oligonucleotides containing d Z and d P were synthesized on an ABI 394 DNA Synthesizer following standard phosphoramidite chemistry and as previously reported (19). The CPGs having oligonucleotides were treated with 2.0 ml of 1 M DBU in anhydrous acetonitrile at room temperature for 24 h to remove the NPE group from the d Z nucleobase.…”

“…These are observed in B-form and A-form, respectively (19–21). However, P:Z pairs impart to the double helix some novel properties (19–21).…”

Xeno-Nukleik Asitler ve XNA Dunyasi

“…Kimoto ve arkadaşları, birbiriyle eşleşebilen ve doğal olmayan iki nükleotidi (Ds:Px) kullanarak DNA replikasyon sırasında DNA'ya üçüncü baz çiftini yerleştirebilmiştir (16). Benzer şekilde Watson-Crick baz eşleşme kurallarına uygun Z:P baz çifti de DNA'ya sokulmuştur (17).…”