Analytical consideration of the selectivity of oligonucleotide hybridization

Systematic analysis of factors determining efficiency in discrimination of a point substitution (SNP) within specific DNA sequences was carried out in the context of hybridization approach. There are two types of selectivity that are critical for the rational design of highly specific oligonucleotides probes. The first type is the real selectivity of hybridization (fa) that is the ratio of association degrees of targets with an oligonucleotide probe upon the perfect and imperfect complex formation. This type of selectivity reflects the level of discrimination between matched and mismatched signals, which is determined both by experimental conditions and the thermodynamics of oligonucleotide hybridization. The second parameter characterizing the efficiency of SNP discrimination is the limit selectivity of hybridization, which determines the utmost value of fa at a given temperature. This value can be calculated as the ratio of corresponding equilibrium association constants of perfect and imperfect complex formation determined purely by thermodynamics. We have shown that the fa function is the most reliable characteristic describing the hybridization selectivity. For the analytical system designed to reveal any type of perturbation in DNA (e.g. SNP or modification), there is usually a temperature at which fa has its maximum value. The dependency of the fa maximum on different experimental parameters as well as the structural characteristics of a probe are described in details. The results allowed us to postulate points of principle to rationally design the most selective probes on the basis of oli- gonucleotides or their derivatives.

Combined effects of oncolytic vaccinia virus and dendritic cells on the progression of melanoma B16-F10 in mice

Aim:We aimed to test the hypothesis that loading of dendritic cells(DCs)with both viral and tumor-specific antigens would enhance the efficacy antitumor DC-based therapy applied simultaneously with oncolytic virus.Methods:Vaccinia virus LIVP/GFP and melanoma B16-F10 were used in this study.DCs were pulsed with various combinations of viral and tumor-associated antigens.The maturation status of DCs was verified by expression of the markers CD80,CD86,and CCR7 and assessment of IL-6,TNF-α,and IL-12 secretion.The most efficient combination of antigens for DC loading was selected based on the analysis of the cytotoxic activity of T lymphocytes.Combination therapy using vaccinia virus LIVP/GFP and DCs pulsed with viral and tumor-specific antigens was administered to the B16-F10 melanoma/mouse C57Bl tumor model.Results:We found that loading of DCs with viral antigens,or with a combination of viral and tumor antigens,resulted in similar levels of expression of DC maturation markers.The maximal in vitro cytotoxicity against virus-infected and non-infected B16 melanoma cells exhibited T lymphocytes activated by DCs loaded with the heat inactivated lysate of vaccinia virus LIVP/GFP infected tumor cell.The results show that the combination of vaccinia virus LIVP/GFP and DCs loaded with both tumor and viral antigens inhibit tumor growth of B16-F10 murine melanoma by more than two-fold.Conclusions:Combination therapy with oncolytic vaccinia virus LIVP/GFP and tumor/virus antigen-loaded DCs limited the growth of established melanoma B16-F10,but no synergistic antitumor effects were observed.We propose that optimization of the therapy regimen could enhance the efficiency of combination therapy.