Interestingly, some β-like DNA Polymerases are able to carry out non-template synthesis of short (up to 300 n) polydeoxyribonucleotides under the conditions of supersaturation with nucleoside triphosphates (50 mM - 200 mM) in the incubation medium [34] [47] [48] . Although the mechanism of this phenomenon is still unclear, the process seems alike to the 3’-terminal polyadenylation of mRNA precursors [49] . The magnetic isotope effect (MIE) of ²⁵Mg²⁺ cations in the cytoplasm of tumor cells (HL-60, WERI-1A, and Y-79) can manifest in the hyperproduction of ATP due to a direct impact on the functioning of nucleotidyl kinases (creatine kinase, pyruvate kinase, etc.), thus creating conditions for supersaturating the intranuclear pool with 2’-deoxyribonucleotide triphosphates (dNTPs) and, accordingly, for initiating non-template polymerization of the latter. This, in turn, contributes to a cytostatic effect due to the decreased efficiency of DNA repair [15] [50] - [52] .

As seen from above, very few of the β-like polymerases ever tested are indeed capable of being promoting such an unusual, merely “abnormal”, catalytic behavior [15] [47] - [52] . It looks like this depends on unique structural peculiarities—3D simplicity [6] [12] [53] and the high ionic strength (4.0 - 8.5 SSC) catalytic resistance [13] [54] in vitro. Noteworthy, despite of their obvious chemical-enzymological significance, these findings are meaningless in terms of the in vivo related scenaria. Taking into account an extremely high level of dNTP concentration required, it is hardly possible to assume any physiologically realistic situation leading to a non-template DNA fragments formation [13] [53] [55] . So there is no a reliable pharmacological potential beyond.

As was mentioned above, in the cells of malignant tumors, the overexpression of β-like DNA Polymerases leads to the increased biosynthesis rate of these enzymes. As a result, DNA is resistant to damage (mutations), and tumor cells acquire high viability and proliferative potential. In cancers, the limited efficiency of DNA repair (BER) mediated by polβ predetermines genome instability [56] . That is why this type of DNA Polymerases is considered as a target for anti-cancer drugs [44] [52] [56] - [59] . In this regard, the search for the effective inhibitors of these enzymes or the suppressors of their synthesis seems to be a promising area of pharmacological exploration.

To block DNA repair, antimetabolites, such as the various derivatives (analogues) of dNTP [15] , can be used as polβ inhibitors, along with the irreversible ones [56] [59] - [61] . However, the high cytotoxicity of such inhibitors limits the possibility of their clinical application. At the same time, the paramagnetic cations of divalent metals are promising inhibitors of β-like DNA Polymerases [46] [62] [63] .

A highly selective inhibitory effect of spinless paramagnetic nuclei of some metal isotopes on catalytic activity of β-like DNA Polymerases is based on the MIE. The replacement of the “non-magnetic” magnesium isotope in the active center of the enzyme by ²⁵Mg²⁺ decreases the enzyme’s activity, which manifests in the reduced rate of ssDNA synthesis and length in vivo. As a result, the contribution of β-like DNA Polymerases to DNA repair in tumor cells is limited, thereby reducing their proliferative activity and viability. The inhibitory effect of the paramagnetic isotopes of ²⁵Mg²⁺, ⁴³Ca²⁺, and ⁶⁷Zn²⁺ on β-like DNA Polymerases was demonstrated on human acute myeloid leukemia HL-60 cells and retinoblastoma cells [15] [46] [64] - [66] .

The decline in the activity of these enzymes naturally leads to a reduced efficiency of their functioning. In the presence of the paramagnetic cation in the incubation medium the enzyme synthesizes shorter ssDNA fragments [46] [66] [67] . However, the inhibitory effect of the paramagnetic divalent cations on β-like DNA Polymerases can be sharply reduced by increased Fe²⁺ concentration in the medium. As a result, the action of such cations is limited in tissues rich in endogenous iron. For instance, in the cells of liver and spleen in mammals, the inhibitory effect of the paramagnetic cations does not appear at all [68] .

As stated above, this is the MIE which underlies the effect of the paramagnetic ²⁵Mg²⁺ cation on β-like DNA Polymerases. The enzymatic process of the addition of a 2’-deoxyribonucleotide residue to a polynucleotide chain occurs not only via the classical reaction of nucleophilic substitution, but can also be associated with the formation of a radical ion intermediate. In the formation of the latter the magnesium cation is involved ( Figure 2 ) [62] . In this process, an electron from 3’O⁻ within the 2’-deoxyribose residue is transferred to the metal cation. This is a key step in the DNA synthesis, resulting in the formation of a radical-ion pair [3’O^. and Mg⁺] and further attachment of the oxyradical to the double bond of Рα = O 2’-deoxyribonucleotide triphosphate. With that, a pyrophosphate is released. It is the participation of the magnesium cation in the reaction catalyzed by polβ, coupled with the radical ion mechanism, that determines the possibility of MIE formation and, thus, the “spin-sensitive” nature of this process [15] [69] .

Besides, the reaction catalyzed by β-like DNA Polymerases can be modulated by the external magnetic field. In the studies on HL-60 cells, the synthesis of single-stranded polynucleotide molecules by β-like DNA Polymerases was shown to be inhibited under the influence of the external magnetic field with 1000 - 1500 H inductance. Furthermore, the presence of a paramagnetic magnesium cation (²⁵Mg²⁺) in the incubation medium significantly enhanced this inhibitory effect [53] [69] .

A mechanism is proposed to explain this phenomenon. According to [30] [69] , an interaction occurs between the “magnetic” nuclei of the divalent metal, which act as electron acceptors forming a radical-ion pair with the oxygen of a phosphate group (electron donor) due to an ultrafine Coulomb effect on the paramagnetic domain. In the process, the “magnetic” cation induces a singlet-triplet transition of the radical-ion pair [12] . The ion-radical intermediate that appears during the reaction can then easily recombine to form the initial reactants or undergoes ST-conversion. As the rate of the ST-conversion increases, the rate of the enzymatic reaction increases as well. Therefore, that very stage of the reaction is “spin-sensitive”. Moreover, when the non-magnetic ²⁴Mg²⁺ is replaced by the paramagnetic ²⁵Mg²⁺ and/or under the influence of an external magnetic field of a certain inductance, the rate of the ssDNA synthesis by β-like DNA Polymerases slows down [13] [53] .

As a result of the inhibitory effect of the paramagnetic cations on β-like DNA Polymerases, the single-stranded polynucleotides synthesized become much shorter than normal ones and consist of only 40 - 100 2’-deoxyribonucleotide residues [46] . This is not enough for the full process of DNA repair in tumor cells [62] . Besides, the short ssDNA fragments also possess inhibitory properties, thus enhancing the inhibitory effect of the paramagnetic cations.

In this way, by inhibiting the activity of β-like DNA Polymerases, ²⁵Mg²⁺ contributes to the antitumor effect [12] [15] [17] [25] [30] [33] [43] - [46] [63] [64] [66] - [71] .

The pharmacological potential of the paramagnetic isotopes of the divalent metal cations (²⁵Mg²⁺, ⁴³Ca²⁺, and ⁶⁷Zn²⁺) as cytostatic agents targeting β-like DNA Polymerases can be applied for anticancer therapy, though the certain data describe below should be taken into account [6] [11] [13] [53] [55] .

First, there is an issue of the targeted delivery of the paramagnetic ions to the tumor cells and, accordingly, the selectivity of their accumulation in the “center of malignancy” [15] [50] [72] [73] . This can be achieved both through the use of porphyrin-fullerene cation-exchange nanoparticles of the PMC16 family ( Figure 3 ) [15] [50] and through “non-Markovian discrimination”. The PMC16 nanoparticles have high affinity to porphyrin-signaling proteins on the mitochondria outer membranes of lymphoblasts, promyelocytes, and acute myeloblastic leukemia cells [73] [74] . The “non-Markovian discrimination” means the preferential accumulation of the amphiphilic nanoparticles (such as PMC16) in the intensively growing tumor tissue (“expanding reservoir”), compared to the neighboring area of the normal tissue not characterized by invasive growth [72] [75] .

The second issue is the mobility of chromatin, which limits the availability of the targets from among its protein components, such as β-like DNA Polymerases. This, in turn, contributes to the selectivity of the cation-protein interaction, which occurs during a short interphase typical for most tumor cells [76] [77] .

In a majority of the DNA polß-devoted works, a non-monomeric subunits possessing structure does not fit a MIE-centered hypothesis of a nuclear spin selective suppression of magnesium-dependent DNA repair in malignant tumors [3] - [6] [8] - [10] . This, nonetheless, is in a good favor with some DFT and X-ray crystallographic models showing that only a relatively small amounts of enzyme species, β-like ones, characterizes by “unusually short” (5.0 - 7.0 nm) distance between the catalytic site coordinated magnesium ion (electron acceptor) and the phosphate oxygen (electron donor) which is a critical condition for Coulomb-related singlet-triplet conversion within a resulted ion-radical pair [7] [13] [53] . That explains a high selectivity of some β-like Polymerases as the MIE participants and therefore, as a potential targets for paramagnetic ion-cytostatics (²⁵Mg²⁺, ⁴³Ca²⁺, ⁶⁷Zn²⁺) [7] [11] [53] [78] .

As follows from a number of researchers, the amphiphilic nanocationites based on porphyrin adducts of fullerene-C₆₀ [51] [75] [79] and carboxymethyl hydroxyapatite [51] [74] may serve as promising pharmacophores that meet the criteria for ensuring targeted in vivo delivery of ²⁵Mg²⁺, ⁴³Ca²⁺, and ⁶⁷Zn²⁺ cations into the cells of human tumors transplanted into animals, such as B16 melanoma, P388 leukemia, and LLC 27 (Lewis lung carcinoma) [7] [55] .

Thus, nanocationites based on porphyrin-fullerenes (РМС16, Figure 3 ) are used for targeted delivery of ²⁵Mg²⁺ into tumor cells for the inhibition of β-like DNA Polymerases [15] . Each of the particles is capable of simultaneously transporting up to 4 divalent cations. Porphyrin-binding signaling proteins of the outer mitochondrial membranes of myeloblasts, promyelocytes, and a number of other cells serve as cation receptors [80] . At the same time, the release of the transported cation from the nanocontainer occurs only under the conditions of metabolic acidosis, which is specific for tumor tissue. Such a method of delivery may be especially promising for the treatment of malignant tumor metastases [15] [81] .

Taking into account the promising potential of the paramagnetic isotope of the magnesium cation as an inhibitor of β-like DNA Polymerases in malignant tumors, the possibility of its uncontrolled negative effect on numerous metalloenzymes, including those of healthy cells, should be evaluated. Estimating this probability, it should be noted that most of the known metal-containing eukaryotic enzymes involved in the processes of intermolecular phosphate transfer have structural features that do not allow them to realize the magnetic isotope effect and, therefore, exclude the participation of these enzymes in a chaotic non-selective response to the presence of ²⁵Mg²⁺, ⁴³Ca²⁺, and ⁶⁷Zn²⁺cations [15] [50] [51] [76] [77] . The possible explanation for this phenomenon is that, according to the nanotopology of their active sites, the distance between the electron donor (the oxygen atom of the transferred phosphate group) and its acceptor (the metal cation) exceeds the distance of 7 - 10 nm, critical for the Coulomb ultrafine induction of singlet-triplet conversion of ion-radical pairs [51] [77] [79] . Such enzymes, in contrast to β-like DNA Polymerases, cannot serve as targets for paramagnetic cytostatic cations. The list of similar enzymes incapable of participating in spin-selective catalysis is very large [15] [75] [79] .

Thus, the small number and relative homogeneity of the enzymes subjected to the effects of the magnetic isotopes (such as β-like DNA Polymerases from leukemia and retinoblastoma cells) are among the factors determining the selectivity of these agents as cytostatic drugs [7] [11] [78] .

The aptamer-like ultra short (30 – 150 n) single-stranded DNA fragments were detected in the blood of many cancer patients [82] [83] . For instance, patients with retinoblastoma demonstrate ultrashort single-stranded polydeoxyribonucleotides consisting from 50 - 150 nucleotide residues in their blood, not detected in healthy donors [70] . Such ultrashort circulating ssDNA fragments can be released into the blood from tumor cells during the repair of their genome.

The polynucleotide chains consisting of 40 - 100 of 2’-deoxyribonucleotides residues demonstrate inhibitory properties towards β-like DNA Polymerases from the malignant tumor cells of HL-60, WERI-RB-1, and Y-79 at the concentration of 6 - 60 μg/ml in the medium [33] [62] . At the same time, a positive correlation was found between the strength of the inhibitory effect and the affinity of the enzyme to the ligand. Interestingly, the inhibitory effect strength relies on the length of ssDNA rather than on its nucleotide composition. The maximum inhibitory effect was established for polynucleotides consisting of 40 - 60 2’-deoxyribonucleotide residues [33] [44] .

The inhibitory effect of short ssDNA chains is explained by their reversible binding to the active site of the enzyme via van der Waals interactions. This process is nonspecific, and the inhibitor binding efficiency directly depends on the length of the polynucleotide chain [44] [71] .

The short polynucleotides were shown to inhibit the reparative DNA Polymerases (such as polβ), and do not affect the replicative ones [33] .

Based on the inhibitory effect of short and ultrashort polynucleotide chains on β-like DNA Polymerases and the fact that ssDNA easily penetrates the intracellular compartments (nuclei and mitochondria) [54] , they can be used in tumor chemotherapy [33] [43] [44] [62] .

The antitumor effect of 2’-deoxypolyribonucleotides was established both in tumor cell cultures and in the experiments on animals with various tumors (B16 melanoma, lung carcinoma, and P388 lymphoid leukemia) [33] . However, the practical use of the short polynucleotides in oncology is limited by the difficulty of their injection into tumor tissue. At the same time, special nanocarriers have recently been proposed for their targeted delivery to a tumor [15] [33] [84] [85] .

Also, the use of L-polydeoxyribonucleotides eliminates their destruction by nucleases, as they hydrolyze only those polydeoxyribonucleotides which consist of D-monomers. Moreover, polynucleotides consisting of L-2’-deoxyribonucleotides were shown to have a more pronounced inhibitory effect towards β-like DNA Polymerases in human acute myeloid leukemia cells [34] [44] [71] .