Aptamers are short single-stranded oligonucleotides that are capable of binding various molecules with high affinity and specificity. When the technology of aptamer selection was developed almost a quarter of a century ago, a suggestion was immediately put forward that it might be a revolutionary start into solving many problems associated with diagnostics and the therapy of diseases. However, multiple attempts to use aptamers in practice, although sometimes successful, have been generally much less efficient than had been expected initially. This review is mostly devoted not to the successful use of aptamers but to the problems impeding the widespread application of aptamers in diagnostics and therapy, as well as to approaches that could considerably expand the range of aptamer application.

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DNA polymerase iota (Pol iota) of mammals is a member of the Y family of DNA polymerases. Among many other genome caretakers, these enzymes are responsible for maintaining genome stability. The members of the Y-family DNA polymerases take part in translesion DNA synthesis, bypassing some DNA lesions, and are characterized by low fidelity of DNA synthesis. A unique ability of Pol iota to predominantly incorporate G opposite T allowed us to identify the product of this enzyme among those synthesized by other DNA polymerases. This product can be called a "false note" of Pol iota. We measured the enzyme activity of Pol iota in crude extracts of cells from different organs of five inbred strains of mice (N3H/Sn, 101/H, C57BL/6, BALB/c, 129/J) that differed in a number of parameters. The "false note" of Pol iota was clearly sounding only in the extracts of testis and brain cells from four analyzed strains: N3H/Sn, 101/H, C57BL/6, BALB/c. In mice of 129/J strain that had a nonsense mutation in the second exon of the pol iota gene, the Pol iota activity was reliably detectable only in the extracts of brain. The data show that the active enzyme can be formed in some cell types even if they carry a nonsense mutation in the pol iota gene. This supports tissue-specific regulation of pol iota gene expression through alternative splicing. A semiquantitative determination of pol iota activity in mice strains different in their radiosensitivity suggests a reciprocal correlation between the enzyme activity of pol iota in testis and the resistance of mice to radiation.

Mammalian Pol ι has an unusual combination of properties: it is stimulated by Mn2+ ions, can bypass some DNA lesions and misincorporates “G” opposite template “T” more frequently than incorporates the correct “A.” We recently proposed a method of detection of Pol ι activity in animal cell extracts, based on primer extension opposite the template T with a high concentration of only two nucleotides, dGTP and dATP (incorporation of “G” versus “A” method of Gening, abbreviated as “misGvA”). We provide unambiguous proof of the “misGvA” approach concept and extend the applicability of the method for the studies of variants of Pol ι in the yeast model system with different cation cofactors. We produced human Pol ι in baker's yeast, which do not have a POLI ortholog. The “misGvA” activity is absent in cell extracts containing an empty vector, or producing catalytically dead Pol ι, or Pol ι lacking exon 2, but is robust in the strain producing wild-type Pol ι or its catalytic core, or protein with the active center L62I mutant. The signature pattern of primer extension products resulting from inaccurate DNA synthesis by extracts of cells producing either Pol ι or human Pol η is different. The DNA sequence of the template is critical for the detection of the infidelity of DNA synthesis attributed to DNA Pol ι. The primer/template and composition of the exogenous DNA precursor pool can be adapted to monitor replication fidelity in cell extracts expressing various error-prone Pols or mutator variants of accurate Pols. Finally, we demonstrate that the mutation rates in yeast strains producing human DNA Pols ι and η are not elevated over the control strain, despite highly inaccurate DNA synthesis by their extracts.

The recently discovered DNA polymerase iota differs greatly from the numerous eukaryotic and prokaryotic DNA polymerases known previously in its ability to catalyze error-prone DNA synthesis. Using homogeneous preparations of the enzyme, it was shown previously that DNA polymerase iota incorporated preferentially dGMP opposite the thymidine of the template in the growing DNA chain. To elucidate the role of this enzyme in the mammals, its activity was assayed in crude cell extracts of different mouse organs. It is shown that the extracts of the brain and testis cells exhibit the highest activity of DNA polymerase iota, which is not in agreement with the results of other authors. The data suggest that the tissue specific expression of DNA polymerase iota is regulated to a significant degree at the posttranscriptional and posttranslational levels.

DNA polymerase β (polβ), a member of the X family of DNA polymerases, is the major polymerase in the base excision repair pathway. Using in vitro selection, we obtained RNA aptamers for polβ from a variable pool of 8 × 1012 individual RNA sequences containing 30 random nucleotides. A total of 60 individual clones selected after seven rounds were screened for the ability to inhibit polβ activity. All of the inhibitory aptamers analyzed have a predicted tri-lobed structure. Gel mobility shift assays demonstrate that the aptamers can displace the DNA substrate from the polβ active site. Inhibition by the aptamers is not polymerase specific; inhibitors of polβ also inhibited DNA polymerase κ, a Y-family DNA polymerase. However, the RNA aptamers did not inhibit the Klenow fragment of DNA polymerase I and only had a minor effect on RB69 DNA polymerase activity. Polβ and κ, despite sharing little sequence similarity and belonging to different DNA polymerase families, have similarly open active sites and relatively few interactions with their DNA substrates. This may allow the aptamers to bind and inhibit polymerase activity. RNA aptamers with inhibitory properties may be useful in modulating DNA polymerase actvity in cells.

An increased level of mutagenesis, partially caused by imbalanced activities of error prone DNA polymerases, is a key symptom of cell malignancy. To clarify the possible role of incorrect DNA polymerase iota (Pol iota) function in increased frequency of mutations in mammalian cells, the activity of this enzyme in extracts of cells of different mouse organs and human eye (melanoma) and eyelid (basal-cell skin carcinoma) tumor cells was studied. Both Mg2+, considered as the main activator of the enzyme reaction of in vivo DNA replication, and Mn2+, that activates homogeneous Pol iota preparations in experiments in vitro more efficiently compared to all other bivalent cations, were used as cofactors of the DNA polymerase reaction in these experiments. In the presence of Mg2+, the enzyme was active only in cell extracts of mouse testicles and brain, whereas in the presence of Mn2+ the activity of Pol iota was found in all studied normal mouse organs. It was found that in cell extracts of both types of malignant tumors (basal-cell carcinoma and melanoma) Pol iota activity was observed in the presence of either Mn2+ or Mg2+. Manganese ions activated Pol iota in both cases, though to a different extent. In the presence of Mn2+ the Pol iota activity in the basal-cell carcinoma exceeded 2.5-fold that in control cells (benign tumors from the same eyelid region). In extracts of melanoma cells in the presence of either cation, the level of the enzyme activity was approximately equal to that in extracts of cells of surrounding tumor-free tissues as well as in eyes removed after traumas. The distinctive feature of tissue malignancy (in basal-cell carcinoma and in melanoma) was the change in DNA synthesis revealed as Mn2+-activated continuation of DNA synthesis after incorrect incorporation of dG opposite dT in the template by Pol iota. Among cell extracts of different normal mouse organs, only those of testicles exhibited a similar feature. This similarity can be explained by cell division blocking that occurs in all normal cells except in testicles and in malignant cells.

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