Discriminator nucleotide

Amexis and coworkers reported an interesting application of semi-quantitative analysis [217] when they employed the MALDI-TOF-MS analysis of primer extension products in their vaccine quahty control process. The ratios of viral quasispecies of the mumps vims were determined between Jeryl Lynn substrains in live, attenuated mumps/measles vaccine. Determination of the ratio of two substrains was performed at five discriminative nucleotide positions within the viral genome. Methods such as this are important for maintaining vaccine safety. 

Figure 10.12 Response elements for heterodimers of the nuclear receptor for ds-retinoic acid (RXR) with the receptors for vitamin D (VDR), thyroid hormone (TR) and trans-retinoic acid (RAR). The half-sites of these response elements have identical nucleotide sequences and are organized as direct repeats. They differ in the number of base pairs in the spacer region between the half-sites. This difference forms the basis for the ability of the heterodimers to discriminate between the different response elements.

Nucleoside and nucleotide reverse transcriptase analogues (NRTI) lack a 3 hydroxyl group and as a result no additional nucleotides can be incorporated into the growing DNA chain. Two NRTI resistance mechanisms are identified impairment of the incorporation of the antiretroviral drug (discrimination) and removal of the analogue from the terminated DNA chain (excision) as reviewed in Chap. 3 (Arion et al. 1998 Meyer et al. 1999 Saralianos et al. 1999). 

Considerable amounts of quenching of the acridone emissions by guanine in the DNA occurred when guanine was close to acridone, which can be applied as a quencher-free probe (no additional quencher is required) for the detection of a special sequence of DNA. The DNA bearing acridone at the C5 position of inner thymidine could distinguish the opposite T-T base mismatch, while enhancement of discrimination ability is needed for the practical use of single nucleotide polymorphism (SNP) typing. 

Another hypothesis was provided by Mikio Shimitso (1982) on the basis of studies of steric effects in molecular models. It had been noted years previously that the fourth nucleotide at the 3 end of the tRNA molecules (referred to as the discrimination base) might have a recognition function. In the case of certain amino acids (i.e., their tRNA-amino acid complexes) this base pair, in combination with the anticodon of the tRNA molecule, can select the amino acid corresponding to the tRNA species in question this is done on the basis of the stereochemical properties of the molecule. Since the anticodon of a tRNA molecule and the fourth nucleotide of the acceptor stem are far apart in space, two tRNA molecules must complex in a head-to-tail manner. The pocket thus formed can then fit specifically to the corresponding amino acid. 

To date, D coefficients of carbohydrates established with the PFGSE approactf - " have been undertaken to (1) validate the theoretical self-diffusion coefficients calculated from MD trajectories, (2) demonstrate the complexation of lanthanide cations by sugars,(3) probe the geometry of a molecular capsule formed by electrostatic interactions between oppositely charged P-cyclodextrins, (4) study the influence of concentration and temperature dependence on the hydrodynamic properties of disaccharides, and (5) discriminate between extended and folded conformations of nucleotide-sugars. ... 

The macrocyclic polyamines, in their multiply-protonated polycationic forms, displayed potentiometric discrimination of adenine nucleotides or dicarboxy-... 

From Table 8 it is obvious that the resolution always increases with an increase of the number of benzene rings and that riboflavine is a more powerful selector than the nucleotides, but not as good as TAPA. An interesting experiment shows that it is not always necessary to have the selector coated or bound to the solid phase but that it can sometimes be used as well, dissolved in the mobile phase. The n-dodecyl ester of N-(2,4-dinitrophenyl)-L-alanine is able to discriminate between the enantiomers of l-aza-[6]-helicene, when used as a chiral dopant in the mobile phase in HPLC on a reversed phase column 93) (see Table 9). The usefulness of this dopant must be due to the known ability of a dinitrophenyl moiety to form CT-complexes with polycyclic aromatic hydrocarbons the presence of a chiral site near this group causes resolution of helicenes, because the steric interactions in diastereomeric complexes will be quite different. 

The structure determination confirmed the importance of the spacing of the two hexamers as a discrimination factor in an impressive maimer. A spacing of only 3 nucleotides between the two hexamers would lead to steric overlap of both receptors a high affinity, cooperative binding would not be possible. With a spacing of more than 4 nucleotides a high affinity complex could also not be formed due to the relative rigidity of the two monomers. 

Around 1980, Tabushi and coworkers used the lipophilized, DABCO-derived diammonium salt 52 as a phase transfer reagent for the transport of nucleotides in three-phase experiments (H2O-CHCI3-H2O) [75]. Whereas AMP was discriminated in a high degree, for ADP and ATP the acceleration rates were quite similar. The transport rates were significantly diminished for uracil and guanine nucleotides because of the low solubility of the resulting complexes. 

Figure 10.15 Electrochemical discrimination of single-nucleotide mismatch with Fc-ODN. (a) probe hybridized to its complementary strand, (b) probe hybridized to single-nucleotide mismatched strand, and center chemical structure of the HS-DNA-Fc probe. Reprinted with permission from Ref. 109. Copyright 2005 National Academy of Sciences, USA.

Figure 27-16 summarizes what we know about the nucleotides involved in recognition by some aminoacyl-tRNA synthetases. Some nucleotides are conserved in all tRNAs and therefore cannot be used for discrimination. 

FIGURE 27-16 Nucleotide positions in tRNAs that are recognized by aminoacyl-tRNA synthetases. Some positions (blue dots) are the same in all tRNAs and therefore cannot be used to discriminate one from another. Other positions are known recognition points for one (orange) or more (green) aminoacyl-tRNA synthetases. Structural features other than sequence are important for recognition by some of the synthetases. 

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