Reaction of homopyrimidine oligonucleotides bearing a 5'-terminal alkylating aromatic 2-chloroethyl-amino group with a bovine papilloma vector expressing human interferon-gamma was investigated. The oligonucleotide derivatives bound to corresponding homopurine-homopyrimidine sequences in dsDNA and alkylated guanosine residues at these sites in the purine strand of the target. The alkylated DNA can be cleaved at the modified residues. At pH 5.4, the reaction was highly specific to the target sequences; at pH less than 5, some nonspecific reactions were observed at the sequences partially complementary to the oligonucleotides. Elongation of the linker between the alkylating group and the oligonucleotide phosphate increased the alkylation efficiency. Repeated treatment of the DNA with gradually increased concentrations of the reagent resulted in quantitative modification of the target guanosines.

We used a DNA duplex formed between the 5' end of a 69mer (69T) and an 11mer (OL7) as a substrate for BamHI. The former oligonucleotide folds into a hairpin structure, the stem of which contains a stretch of pyrimidines in one strand and consequently a stretch of purines in the other strand. The oligomer 69T was used as a target for complementary oligodeoxypyrimidines made of 10 nt (OL1), 16 nt (OL5) or 26 nt (OL2) which can engage the same 10 pyrimidine-purine-pyrimidine triplets with the 69T hairpin stem. Although the binding site of OL7 did not overlap that of OL1, OL2 or OL5, the BamHI activity on 69T-OL7 complexes was drastically modified in the presence of these triplex-forming oligomers: OL1 abolished the cleavage by BamHI whereas OL5 and OL2 strongly increased it. Using footprinting assays and point-mutated oligonucleotides we demonstrated that these variations were due to different conformations of the 69T-OL7 complex induced by the binding of oligomers OL1, OL2 or OL5. Therefore, oligonucleotides can act as structural switchers, offering one additional mode for modulating gene expression.

Sequence-specific alkylation of dsDNA with pyrimidine oligonucleotides bearing an alkylating group at the 3' and 5' terminal phosphates, or both, has been investigated. At pH 5.4, sequence-specific modification of guanosines of the DNA in the vicinity of the target purine-pyrimidine sequence occurs. The reactive group at the 5' terminus of the oligonucleotides attacks guanosines in the purine strand of the target DNA. The reactive group at the 3' end can interact with guanosines in both strands of the DNA. Bifunctional reagents can alkylate both strands of the DNA simultaneously. At pH 4 in the presence of magnesium ions, the oligonucleotide derivatives can form imperfect complexes with sequences homologous to the target sequence and alkylate the DNA at the corresponding positions.

The clinical course of active retinopathy after anti-VEGF therapy, the possibility and timing of recurrence of the disease, anatomical and functional outcomes of treatment are widely discussed in the press, not fully studied and relevant.Purpose: to study the clinical course of active retinopathy of prematurity after anti-VEGF therapy and clinical and functional outcomes.Patients and Methods. Children with active retinopathy of prematurity, who turned to the Helmgoltz National Medical Research Centre of Eye Diseases after anti-VEGF therapy, examined by indirect binocular ophthalmoscopy and digital retinal camera (RetcamShuttle). Children were monitored from 1.5 to 6 years (average 2.94 ± 1.47). All children underwent routine examination, 4 children older than 3 years underwent optical coherence tomography.Results. In all cases, after anti-VEGF therapy, there was a decrease in vascular activity and continued vascularization of the retina. Recurrence of the disease requiring additional treatment, were detected in 11 (42.3 %) eyes within 6–22 weeks (in average 13.33 ± 5.57) after intravitreal anti-VEGF therapy. Laser coagulation of the retina was carried out in 4 children (7 eyes) and repeated administration of anti-VEGF drug — 2 children (4 eyes), which led to regression of the disease. In the long-term period, all 13 (100 %) children had successful outcomes.Conclusion. Anti-VEGF therapy is effective in plus-zone 1 disease and posterior aggressive retinopathy of prematurity. Its advantages include the ability to treat retinopathy in zone 1 posterior, the absence of “blockade” of the peripheral retina with the possibility of continued growth of blood vessels to the periphery, lower frequency and severity of myopia. The disadvantages include the possibility of recurrence of the disease, which requires long-term regular monitoring.