Chemical Morphing of DNA Containing Four Noncanonical Bases

Abstract

The ability of alternative nucleic acids, in which all four nucleobases are substituted, to replicate in vitro and to serve as genetic templates in vivo was evaluated. A nucleotide triphosphate set of 5-chloro-2'-deoxyuridine, 7-deaza-2'-deoxyadenosine, 5-fluoro-2'-deoxycytidine, and 7-deaza-2'deoxyguanosine successfully underwent polymerase chain reaction (PCR) amplification using templates of different lengths (57 or 525mer) and Taq or Vent (exo-) DNA polymerases as catalysts. Furthermore, a fully morphed gene encoding a dihydrofolate reductase was generated by PCR using these fully substituted nucleotides and was shown to transform and confer trimethoprim resistance to E. coli. These results demonstrated that fully modified templates were accurately read by the bacterial replication machinery and provide the first example of a long fully modified DNA molecule being functional in vivo.

PCR Amplification of Base‐Modified DNA

“…10× ThermoPol reaction buffer (New England Biolabs) 1 μM DNA template T57 in sterile H 2 O (Integrated DNA Technology; see Table 1 for sequences; store at -20°C) 10 μM PCR primers Cy5-P1 and Cy3-P2 in sterile H 2 O (Integrated DNA Technology, see Table 1 for sequences; store at -20°C) 5000 U/ml Taq DNA polymerase (New England Biolabs; see Table 2 for specifications) 2000 U/ml Vent (exo -) DNA polymerase (New England Biolabs; see Table 2 for specifications) 2 mM dCTP and dGTP mix (New England Biolabs) 2 mM modified A 1 -A 3 (Ax) and T 1 deoxyribonucleoside 5 -triphosphates (synthesis described in Eremeeva et al, 2016aEremeeva et al, , 2016aEremeeva et al, ,b, 2017 (15 × 10 cm), and comb (20-well 1.5 mm thick) Large horizontal electrophoresis system (e.g., Sub-Cell GT, Bio-Rad) UV transilluminator (VWR) No. of tube 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 Taq DNA polymerase Vent (exo-) DNA polymerase NC T T1 NC T T1 5.…”

“…10× ThermoPol reaction buffer (New England Biolabs) 10 nM DNA libraries, Lib25, Lib40, and p-Lib25 (Integrated DNA Technology, see Table 1 for sequences) 10 μM PCR primers, Cy5-P1, p-P1, and Cy3-P3 (Integrated DNA Technology, see Table 1 for sequences) 5000 U/ml Taq DNA polymerase (New England Biolabs; see Table 2 for specifications) 2 mM natural deoxyribonucleoside triphosphate dNTP set (New England Biolabs) 2 mM modified A 1 + T 1 or A 1 with T 1 (mix), G 1 -G 6 (Gx), and C 1 -C 3 (Cx) deoxyribonucleoside 5 -triphosphates (synthesis described in Eremeeva et al, 2016aEremeeva et al, , 2016aEremeeva et al, ,b, 2017; A 1 , G 1 , G 6 , C 1 , and C 3 2 -deoxyribonucleoside 5 -triphosphates are available in Sigma-Aldrich Benelux (G 1 and G 6 ) or Tebu-Bio France (A 1 , C 1 , and C 3 ) 2× denaturing loading buffer (see recipe) 15% denaturing polyacrylamide gel (PAGE, see recipe) 10× Lambda exonuclease reaction buffer (New England Biolabs) 5000 U/ml Lambda exonuclease (New England Biolabs) 6× native loading buffer (see recipe) (B-C) Image of 15% denaturing PAGE with the relative yield of PCR products to natural product formation, with Lib25 (left) and Lib40 (right). Cy3-or Cy5-labeled PCR products are shown in pink or light blue, respectively; average yield from both labeled strands is shown in dark blue.…”

“…10× ThermoPol Reaction Buffer (New England Biolabs) 25 ng/μl DNA plasmid pXEN156 (see Figure 6 and Figure 7 for detailed sequence of pXEN156 plasmid) 10 μM forward (LongFW1, LongFR2) and reverse (LongRV1-LongRV4) PCR primers (Integrated DNA Technology, see Table 1 for sequence) 5000 U/ml Taq DNA polymerase (New England Biolabs; see Table 2 for specification) 2 mM natural deoxyribonucleoside triphosphates dATP, dTTP, dCTP, dGTP, and dNTP set (New England Biolabs) 2 mM modified A 1 , T 1 , G 1 -G 6 (Gx), and C 1 -C 3 (Cx) deoxyribonucleoside 5 -triphosphates [synthesis described in (Eremeeva et al, 2016a(Eremeeva et al, , 2016bb, 2017, A 1 , G 1 , G 6 , C 1 , and C 3 2 -deoxyribonucleoside 5 -triphosphates are available in Sigma-Aldrich Benelux (G 1 and G 6 ) or Tebu-Bio France (A 1 , C 1 , and C 3 )] 6× Purple Gel Loading Dye (New England Biolabs) 1% agarose gel (see recipe):…”

“…These modified DZA structures can possess increased thermal and endonuclease stability (Eremeeva et al, 2016a(Eremeeva et al, , 2016bb, 2017. These modified DZA structures can possess increased thermal and endonuclease stability (Eremeeva et al, 2016a(Eremeeva et al, , 2016bb, 2017.…”

“…It is possible to directly clone DZA fragments into a commercial vector followed by transforming into a bacterial cell, without the need of reverse transcription back to DNA (Eremeeva et al, 2016a(Eremeeva et al, , 2017. For example, the targeted delivery of therapeutic agents (5fluorocytosine) can be achieved.…”