Nucleotides acid-base behavior

The acid-base behavior of nucleotides is dominated by the acidity of the nucleic bases and the phosphate groups (Table 8.5.2). Some remarkable changes in pk values are observed when going from hydrophobic to hydrophilic derivatives (e.g., it is more difficult to protonate the NH2 of adenosine than that of adenine), the acidity of the anomeric pentose proton (typical pk, values 12-13), and the pk 2 value of the phosphate close to 7. At high pH values, the 20H group of ri-bose can also be deprotonated (Shabarova and Bogdanov, 1994). 

Table 3 considers potential reference shifts for base protons. Here there is considerable variability, but still a rough correspondence between shifts for the isolated bases in acid or base solution (column [1]) and average values from a DNA database (column [5]). The exception is the H8 resonance of guanine, which in basic aqueous solution is about 0.4 ppm upheld from its value in nucleotides or DNA structures. We do not yet understand the origin of this behavior. For the present calculations, we have used the isolated bases as references (shown in bold in Table 3), but have increased the reference shift of guanine H8 by 0.34 ppm. 

Zakaria and Brown (Zl) have found that, whereas nucleoside and base retention mechanisms can be adequately explained in terms of solvophobic considerations, nucleotide retention behavior can best be explained in terms of a mixed-mode mechanism. In an acidic mobile phase, it has been observed that ribonucleotides elute in order of increasing negative charge. This elution pattern is atypical for the reversed-phase... 

Importantly, King succeeded in the formation of a Fc-labeled 998-bp (base-pair) construct by PCR using T4 DNA polymerase in the presence of Fcl-dUTP. Incorporation of the redox label shows that Fcl-dUTP is suitable as a substrate for PCR. In contrast, Fc2-dUTP acts predominantly as a terminator in the PCR. The melting behavior of a 37-mer duplex containing five Fcl-dU residues reveals that the labeled nucleotide induces only a modest helix destabilisation, with T ,=71°C for a labeled duplex versus 75°C for the corresponding nonlabeled ds-DNA construct. King reports that the Fc-labeled DNA is detected at femtomolar levels by HPLC using a coulometric detector. Thus, it must be emphasized that the incorporation of the Fc label by PCR and its facile and cost-effective electrochemical detection should promote the use of this technique in nucleic acid analysis and may replace the more costly fluorescence-based detection systems. 

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