¹⁵N-Enriched 5-Fluorocytosine as a Probe for Examining Unusual DNA Structures

Abstract

A new method is presented for the synthesis of oligonucleotides containing 15N-enriched 5-fluorocytosine (FC). Due to the reduced pK of FC, the amino protons of an unpaired FC residue may be observed at lower values of solution pH. The labeled FC residue has been placed as a template base at a model DNA replication fork. The amino protons of the FC residue have been identified in isotope-edited NMR spectra. Data is presented for a template FC residue unpaired, paired with guanine, and mispaired with adenine. These studies demonstrate the utility of labeled FC in examining unusual DNA structures.

Density functional study of the influence of C5 cytosine substitution in base pairs with guanine

“…Our calculations for the same analogs give 6.29 and 6.98, respectively. Sowers references a 1.8 p K a drop between 5-fluoro-C and cytosine,56 while we show a a 2.9 p K a decrease. While the absolute p K a difference is ~2 p K a units, the relative values that we are relying on are within ~1.0 p K a units.…”

A Mutational Analysis of the 8–17 Deoxyribozyme Core

“…The pK a of 5-methyl-cytosine is slightly higher than that of cytosine (w4.3), whereas the pK a of 5-bromo cytosine is about 1.6 units lower. 43 The reported k rel values refer to reactions carried out in 3 mM Mg 2C , but similar effects were obtained in the presence of 3 mM Ca 2C or 3 mM Mn 2C . k rel for the 6-thio G mutant did not change when activity was measured in the presence of 2 mM dithiothreitol or when the pH of the reaction mixture was lowered from 7.4 to 6.8.…”

“…41,42 If a similar role were played by C13 in the 8-17 mechanism, replacement of the C13 base with 5-bromo cytosine should yield a substantial reduction of the cleavage rate. In fact 5-bromo cytosine (due to its low pK a ) 43 is expected to be about 40-fold less efficient than cytosine as a general acid-base catalyst at neutral pH. In contrast to this expectation, an (8-17)cb variant bearing 5-bromo C at position 13 showed an activity only fourfold lower than that of the wild-type enzyme (Figure 4).…”

“…44 Furthermore, since most mutations at positions 12, 15 and 15.0 are relatively well tolerated, these positions are ideal places for introducing spectroscopic probes (e.g. fluorescent or fluorinated bases) 43 to monitor the conformation of 8-17 during different phases of the catalytic process.…”

Comparison of the Structural and Dynamic Effects of 5-Methylcytosine and 5-Chlorocytosine in a CpG Dinucleotide Sequence

“…The pK a of the N 3 position of dC is 4.2, and an electron donating CH 3 substituent in the 5-position increases the pK a to 4.3 (30). In contrast, an electron withdrawing Cl substituent decreases the pK a of Cl dC to 2.7 (30). In Figure 7a, the G10 chemical shift is plotted versus the pK a of the paired cytosine or cytosine analog, and a linear relationship is observed.…”