Nucleoside Analogue Substitutions in the Trinucleotide DNA Template Recognition Sequence 3‘-(CTG)-5‘ and Their Effects on the Activity of Bacteriophage T7 Primase

Abstract

Bacteriophage T7 primase catalyzes the synthesis of the oligoribonucleotides pppACC(C/A) and pppACAC from the single-stranded DNA template sites 3'-d[CTGG(G/T)]-5' and 3'-(CTGTG)-5', respectively. The 3'-terminal deoxycytidine residue is conserved but noncoding. A series of nucleoside analogues have been prepared and incorporated into the conserved 3'-d(CTG)-5' site, and the effects of these analogue templates on T7 primase activity have been examined. The nucleosides employed include a novel pyrimidine derivative, 2-amino-5-(beta-2-deoxy-D-erythro-pentofuranosyl)pyridine (d2APy), whose synthesis is described. Template sites containing d2APy in place of the cryptic dC support oligoribonucleotide synthesis whereas those containing 3-deaza-2'-deoxycytidine (dc(3)C) and 5-methyl-6-oxo-2'-deoxycytidine (dm(5ox)C) substitutions do not, suggesting that the N3 nitrogen of cytidine is used for a critical interaction by the enzyme. Recognition sites containing 4-amino-1-(beta-2-deoxy-D-erythro-pentofuranosyl)-5-methyl-2,6[1H, 3H]-pyrimidione (dm(3)2P) or 2'-deoxyuridine (dU) substitutions for dT support oligoribonucleotide synthesis whereas those containing 5-methyl-4-pyrimidinone 2'-deoxyriboside (d(2H)T) substitutions do not, suggesting the importance of Watson-Crick interactions at this template residue. Template sites containing 7-deaza-2'-deoxyguanosine (dc(7)G) or 2'-deoxyinosine (dI) in place of dG support oligoribonucleotide synthesis. The reduced extent to which dc(7)G is successful within the template suggests a primase-DNA interaction. Inhibition studies suggest that the primase enzyme binds "null" substrates but cannot initiate RNA synthesis.

Mapping out the Relative Influence of Hydrogen and Halogen Bonds in Crystal Structures of a Family of Amide-Substituted Pyridines

“…mp 121− 122 °C). 33 2Pyr Series. The synthesis of N-(pyridin-2-yl)picolinamides was done according to an available literature procedure.…”

DNA Primases

“…No RNA synthesis is supported by short synthetic templates lacking either the cryptic dC or the adjacent dT in the recognition site (25). Templates containing modified dC or dT bases likewise support a lower rate of oligoribonucleotide synthesis than sequences containing the preferred recognition site 5 -GTC-3 (117,118). In particular, templates lacking the N3 nitrogen of the cryptic dC or the N3 nitrogen of dT support little detectable synthesis, highlighting potential interactions between the primase and functional groups involved in Watson-Crick base-pair formation (118).…”

Controlling the Fluorescence of Benzofuran‐Modified Uracil Residues in Oligonucleotides by Triple‐Helix Formation

“…TFO-1 with a propylene linker (C3) recognizes the HPs. To study the triplexes at pH 7.0, 2-aminopyridine nucleoside (Py) [48][49][50] was used instead of deoxycytidine in TFO-1 because the conditions must be acidic for the protonation of cytosine when a TFO containing deoxycytidine is used. [48] Figure S2).…”

“…TFO-1 with a propylene linker (C3) recognizes the HPs. To study the triplexes at pH 7.0, 2-aminopyridine nucleoside (Py) [48][49][50] was used instead of deoxycytidine in TFO-1 because the conditions must be acidic for the protonation of cytosine when a TFO containing deoxycytidine is used. [48] To confirm triplex formations, the melting temperatures (T m ; transition from the triplex state to oligodeoxynucleotides) for HP-U BF or HP-U MBF with TFO-1 were measured: 33 8C for HP-U BF with TFO-1, and 28 8C for HP-U MBF with TFO-1 ( Figure S2).…”