Double-stranded Oligodeoxyribonucleotides Research Articles

Cas-Clover Editing Efficiency and Off-Target Activity in Human Hepatocytes at the KLKB1 Locus

Cutting-edge gene editing holds enormous promise for tackling devastating genetic diseases like hereditary angioedema (HAE). Here, we describe the efficient inactivation of the gene encoding pre-kallikrein, KLKB1, using our proprietary Cas-CLOVER™ high-fidelity nuclease with our non-viral, lipid nanoparticle (LNP) delivery system. Genetic inactivation of KLKB1 is an alternative clinical approach that provides durable relief to both Type I and II HAE. HAE is a rare genetic disease characterized by subcutaneous and submucosal edema, with swelling of the upper respiratory tract posing a life-threatening situation. Type I and II HAE are the most common types and are caused by mutations in the SERPING1 gene, which leads to compromised production or function of the C1 protease inhibitor. Strategies for treatment and prophylaxis include the restoration of C1 inhibitor function, or downstream antagonism of active plasma kallikrein. Safe and effective gene editing of KLKB1 could be a viable alternative for patients not adequately responding to the current standard of care. However, gene editing approaches must demonstrate an exquisitely high level of fidelity for optimal safety. To demonstrate such an approach with Cas-CLOVER, multiple guide RNAs (gRNA) targeting the human KLKB1 gene were screened in human hepatoma cell lines to identify gRNA pairs with optimal editing. Next, we evaluated KLKB1 protein reduction in primary human hepatocytes (PHH) that were incubated with LNPs encapsulating Cas-CLOVER mRNA along with each gRNA pair. Lead candidate gRNAs showed robust KLKB1 editing in a dose-responsive manner, achieving >65% editing and >85% reduction in KLKB1 protein secreted into culture medium at 0.5 ug/mL (EC90). To evaluate Cas-CLOVER off-target activity, oligo incorporation by iGUIDE was carried out by a licensed contract research organization. In this assay, double-stranded oligodeoxyribonucleotides (dsODNs) were co-electroporated with Cas-CLOVER mRNA, along with our lead KLKB1 gRNA pair, in the Huh7 cell line, and candidate off-target sites were identified by Illumina next-generation sequencing. Off-target activity was assessed by amplicon-seq at the eight top sites nominated by iGUIDE. In PHHs treated with 0.5 ug/mL of Cas-CLOVER LNPs, off-target editing was detected in 3/8 sites at very low levels (<0.25%). Remarkably, this low level of off-target editing remained unchanged when PHHs were treated with 10-fold higher concentrations of Cas-CLOVER LNP. For further evaluation of our platform, we sought to determine KLKB1 editing efficiency and fidelity in a mouse model of liver humanization. TK-Nog mice engrafted with PHHs were treated with a single intravenous injection of an LNP formulation co-encapsulating Cas-CLOVER mRNA and our lead KLKB1 gRNA pair. Amplicon-seq analysis demonstrated that 60% of KLKB1 alleles in the liver were edited. Importantly, no off-target editing was detected among the top eight sites identified by iGUIDE, including the three off-target sites validated in cultured PHHs. Next, we evaluated efficacy and tissue specificity of our platform in wild type mice. C57BL/6 male and female mice were dosed with LNP encapsulating Cas-CLOVER mRNA and mouse Klkb1-targeting gRNAs. A single intravenous LNP injection achieved high Klkb1 editing (>50% of haploid genomes) in the liver and >80% reduction in serum pre-kallikrein levels. No Klkb1 editing was detected in gonads. In summary, these results highlight the efficacy and specificity of our high-fidelity Cas-CLOVER gene editing platform that enables targeted and therapeutically relevant kallikrein reduction in a fully non-viral manner. These data provide a promising foundation for the development of a highly specific gene editing therapy for HAE.

An experimental study of the effects of SNPs in the TATA boxes of the GRIN1, ASCL3 and NOS1 genes on interactions with the TATA-binding protein.

The GRIN1, ASCL3, and NOS1 genes are associated with various phenotypes of neuropsychiatric disorders. For instance, these genes contribute to the development of schizophrenia, Alzheimer’s and Parkinson’s diseases, and epilepsy. These genes are also associated with various cancers. For example, ASCL3 is overexpressed in breast cancer, and NOS1, in ovarian cancer cell lines. Based on our findings and literature data, we had previously obtained results suggesting that the single-nucleotide polymorphisms (SNPs) that disrupt erythropoiesis are highly likely to be associated with cognitive and neuropsychiatric disorders in humans. In the present work, using SNP_TATA_Z-tester, we investigated the influence of unannotated SNPs in the TATA boxes of the promoters of the GRIN1, ASCL3, and NOS1 genes (which are involved in neuropsychiatric disorders and cancers) on the interaction of the TATA boxes with the TATA-binding protein (TBP). Double-stranded oligodeoxyribonucleotides identical to the TATA-containing promoter regions of the GRIN1, ASCL3, and NOS1 genes (reference and minor alleles) and recombinant human TBP were employed to study in vitro (by an electrophoretic mobility shift assay) kinetic characteristics of the formation of TBP–TATA complexes and their affinity. It was found, for example, that allele A of rs1402667001 in the GRIN1 promoter increases TBP–TATA affinity 1.4-fold, whereas allele C in the TATA box of the ASCL3 promoter decreases the affinity 1.4-fold. The lifetime of the complexes in both cases decreased by ~20 % due to changes in the rates of association and dissociation of the complexes (ka and kd, respectively). Our experimental results are consistent with the literature showing GRIN1 underexpression in schizophrenic disorders as well as an increased risk of cervical, bladder, and kidney cancers and lymphoma during ASCL3 underexpression. The effect of allele A of the –27G>A SNP (rs1195040887) in the NOS1 promoter is suggestive of an increased risk of ischemic damage to the brain in carriers. A comparison of experimental TBP–TATA affinity values (KD) of wild-type and minor alleles with predicted ones showed that the data correlate well (linear correlation coefficient r = 0.94, p <0.01).

Recognition of mixed-sequence DNA targets using spermine-modified Invader probes.

Double-stranded oligodeoxyribonucleotides with +1 interstrand zipper arrangements of 2'-O-(pyren-1-yl)methyl-RNA monomers are additionally activated for highly specific recognition of mixed-sequence DNA targets upon incorporation of non-nucleotidic spermine bulges.

A real-time study of the interaction of TBP with a TATA box-containing duplex identical to an ancestral or minor allele of human gene LEP or TPI

It is known that only a single-nucleotide substitution (SNP: a single nucleotide polymorphism) in the sequence of a TATA box can influence the affinity of the interaction of TBP with the TATA box and contribute to the pathogenesis of complex hereditary human diseases and sometimes may be a cause of monogenic diseases (for instance, β-thalassemia). In the present work, we studied the interaction of human TBP with a double-stranded oligodeoxyribonucleotide (ODN) 15 or 26 bp long identical to a TATA box of promoters of a real-life human gene, TPI or LEP, and labeled with fluorophores TAMRA and FAM. To analyze the interaction of TBP with a TATA box of an ancestral or minor allele (SNP in the TATA box) in real time, we used the stopped-flow method with detection of a Förster resonance energy transfer (FRET) signal. The nature of the resulting kinetic curves reflecting changes in the FRET signal (and therefore of DNA conformation during the interaction with TBP) pointed to a multistage mechanism of the formation of the TBP complex with the TATA-containing ODN. The results showed that with the increasing concentration and length of the ODN, heterogeneity of conformational changes (taking place during the first second of the interaction with TBP) in DNA also increases. In contrast to the initial nonspecific interaction, the subsequent phases strictly depend on TBP concentration: at the TBP:ODN ratio of 10:1, the velocity of change of the FRET signal increases approximately 100-fold.

The Effect of Dimethyl Sulfoxide on Supercoiled DNA Relaxation Catalyzed by Type I Topoisomerases.

The effects of dimethyl sulfoxide (DMSO) on supercoiled plasmid DNA relaxation catalyzed by two typical type I topoisomerases were investigated in our studies. It is shown that DMSO in a low concentration (less than 20%, v/v) can induce a dose-related enhancement of the relaxation efficiency of Escherichia coli topoisomerase I (type IA). Conversely, obvious inhibitory effect on the activity of calf thymus topoisomerase I (type IB) was observed when the same concentration of DMSO is used. In addition, our studies demonstrate that 20% DMSO has an ability to reduce the inhibitory effect on EcTopo I, which was induced by double-stranded oligodeoxyribonucleotides while the same effect cannot be found in the case of CtTopo I. Moreover, our AFM examinations suggested that DMSO can change the conformation of negatively supercoiled plasmid by creating some locally loose regions in DNA molecules. Combining all the lines of evidence, we proposed that DMSO enhanced EcTopo I relaxation activity by (1) increasing the single-stranded DNA regions for the activities of EcTopo I in the early and middle stages of the reaction and (2) preventing the formation of double-stranded DNA-enzyme complex in the later stage, which can elevate the effective concentration of the topoisomerase in the reaction solution.

Facile Enzymatic Synthesis of Base J-Containing Oligodeoxyribonucleotides and an Analysis of the Impact of Base J on DNA Replication in Cells

We reported here the use of T4 bacteriophage β-glucosyltransferase (T4 β-GT) for the facile synthesis of base J-containing oligodeoxyribonucleotides (ODNs). We found that the enzyme could catalyze the glucosylation of 5-hydroxymethyl-2-deoxyuridine (5hmU) in both single- and double-stranded ODNs, though the latter reaction occurred only when 5hmU was mispaired with a guanine. In addition, base J blocked moderately DNA replication, but it did not induce mutations during replication in human cells.

Sequence-Selective Detection of Double-Stranded DNA Sequences Using Pyrrole–Imidazole Polyamide Microarrays

We describe a microarray format that can detect double-stranded DNA sequences with a high degree of sequence selectivity. Cyclooctyne-derivatized pyrrole-imidazole polyamides were immobilized on azide-modified glass substrates using microcontact printing and a strain-promoted azide-alkyne cycloaddition (SPAAC) reaction. These polyamide-immobilized substrates selectively detected a seven-base-pair binding site incorporated within a double-stranded oligodeoxyribonucleotide sequence even in the presence of an excess of a sequence with a single-base-pair mismatch.

Ku protein as the main cellular target of cell-surface-bound circulating DNA

Objective: An immunomodulatory activity of circulating DNA (cirDNA) is implemented via the interactions of cirDNA with the targets exposed on the cell membrane and/or intracellular targets. The goal of this work was to identify the cellular targets of immunoinhibiting cell-surface-bound cirDNA (csbDNA) using its oligodeoxyribonucleotide (ODN) analogs containing the nucleotide motifs frequently found in csbDNA and displaying the same effects.Materials and methods: The binding of [32P]-labeled single- and double-stranded ODNs (ss- and ds-ODNs) with membrane–cytosolic (MC) extracts and living human umbilical vein endothelial cells (HUVEC) was studied by electromobility shift assay (EMSA). Complexes of biotinylated ODNs with target proteins were affinity isolated using streptavidin Sepharose with subsequent SDS-PAGE and identified by MALDI-TOF mass spectrometry.Results and conclusions: Both ss- and ds-ODNs form strong ODN–protein complexes with similar electrophoretic mobilities after incubation with the MC extracts of HUVEC either when added extracellularly or lipofected into cells. The ODN-binding proteins were identified as the DNA-binding components of DNA-dependent protein kinase (DNA-PK), namely, Ku70 and Ku80 proteins. Diverse cellular localizations and functions of the Ku proteins demand further clarification of Ku70/80 role as a mediator of the csbDNA immunoinhibiting effects.

Plant mitochondria possess a short-patch base excision DNA repair pathway

Despite constant threat of oxidative damage, sequence drift in mitochondrial and chloroplast DNA usually remains very low in plant species, indicating efficient defense and repair. Whereas the antioxidative defense in the different subcellular compartments is known, the information on DNA repair in plant organelles is still scarce. Focusing on the occurrence of uracil in the DNA, the present work demonstrates that plant mitochondria possess a base excision repair (BER) pathway. In vitro and in organello incision assays of double-stranded oligodeoxyribonucleotides showed that mitochondria isolated from plant cells contain DNA glycosylase activity specific for uracil cleavage. A major proportion of the uracil–DNA glycosylase (UDG) was associated with the membranes, in agreement with the current hypothesis that the DNA is replicated, proofread and repaired in inner membrane-bound nucleoids. Full repair, from uracil excision to thymidine insertion and religation, was obtained in organello following import of a uracil-containing DNA fragment into isolated plant mitochondria. Repair occurred through single nucleotide insertion, which points to short-patch BER. In vivo targeting and in vitro import of GFP fusions showed that the putative UDG encoded by the At3g18 630 locus might be the first enzyme of this mitochondrial pathway in Arabidopsis thaliana.

Interactions of human 8-oxoguanine DNA glycosylase with single-and double-stranded DNAs

Interactions of human 8-oxoguanine-DNA glycosylase (hOGG1) with single- and double-stranded oligodeoxyribonucleotides (ODN) have been studied by the method of stepwise increase in ligand complexity. The ODNs have been found to inhibit the glycosylase-catalyzed reaction competitively. The K1 values have been determined for a set of ODNs. All units of non-specific DNA within the enzyme footprint have been shown to interact with the protein globule in an additive manner. An increase in the d(pN)n length (n) by one unit caused a monotonous 1.4-1.5-fold increase in their affinity for the glycosylase ODN until n = 10, mostly due to weak nonspecific contacts of the enzyme and the sugar-phosphate backbone. The weak nonspecific additive interactions contributed about five orders of magnitude in the affinity of hOGG1 for specific DNA (Kd approximately 10(-5) M), whereas introduction of a 8-oxoguanine residue added about three orders of magnitude to this affinity (Kd approximately 10(-8) M). Quantitative features of recognition of specific DNA by the enzyme are analyzed.

Structure‐based cross‐linking of NF‐κB p50 homodimer and decoy bearing a novel 2'‐disulfide trapping site

Double-stranded oligodeoxyribonucleotides with engineered disulfide units were successfully used for covalent trapping of cysteine containing proteins. In particular, an efficient cross-linking of NF-kappaB p50 homodimer to a sequence-specific decoy was demonstrated. The results suggest that the synthetic oligonucleotides bearing a novel 2'-disulfide trapping site can be used as new tools to study and manipulate biological systems.

The Reactivity of the 5-Hydroxy-5,6-dihydrothymidin-6-yl Radical in Oligodeoxyribonucleotides

Hydroxyl radical can be induced from Fenton reaction or gamma radiolysis of water. It can add preferentially to the C5 position of thymidine to give the 5-hydroxy-5,6-dihydrothymidin-6-yl radical. In this report, we examined the independent generation and reactivity of this radical in oligodeoxyribonucleotides (ODNs). Our results showed that the major products originated from this radical in single- and double-stranded ODNs were thymidine glycol, 5-hydroxy-5,6-dihydrothymidine, thymidine, and abasic site lesion. A cross-linking lesion, where the C6 of 5-hydroxy-5,6-dihydrothymidine and the C8 of its neighboring guanine are covalently bonded, could be induced from the independently generated radical in dinucleoside monophosphate and trinucleoside diphosphates. The formation of this type of cross-link product in duplex ODNs from either the independently generated radical or gamma irradiation was near or below the detection limit of the LC-MS/MS method that we used.

Generation of 5-(2'-deoxycytidyl)methyl radical and the formation of intrastrand cross-link lesions in oligodeoxyribonucleotides

Hydroxyl radical is one of the major reactive oxygen species (ROS) formed from γ-radiolysis of water or Fenton reaction, and it can abstract one hydrogen atom from the methyl carbon atom of thymine and 5-methylcytosine to give the 5-methyl radical of the pyrimidine bases. The latter radical can also be induced from Type-I photo-oxidation process. Here, we examined the reactivity of the independently generated 5-(2′-deoxycytidyl)methyl radical (I) in single- and double-stranded oligodeoxyribonucleotides (ODNs). It was found that an intrastrand cross-link lesion, in which the methyl carbon atom of 5-methylcytosine and the C8 carbon atom of guanine are covalently bonded, could be formed from the independently generated radical at both GmC and mCG sites, with the yield being much higher at the former site. We also showed by LC-MS/MS that the same cross-link lesions were formed in mC-containing duplex ODNs upon γ irradiation under both aerobic and anaerobic conditions, and the yield was ∼10-fold higher under the latter conditions. The independently generated radical allows for the availability of pure, sufficient and well-characterized intrastrand cross-link lesion-bearing ODN substrates for future biochemical and biophysical characterizations. This was also the first demonstration that the coupling of radical I with its 5′ neighboring guanine can occur in the presence of molecular oxygen, suggesting that the formation of this and other types of intrastrand cross-link lesions might have important implications in the cytotoxic effects of ROS.

The binding of zinc (II) to a double-stranded oligodeoxyribonucleotide. A voltammetric study

Binding of zinc to a 19 mer double-stranded oligodeoxyribonucleotide was investigated by anodic stripping voltammetry and cyclic voltammetry in order to understand the roles of zinc in DNA cleavage catalyzed by mung bean nuclease. These methods rely on the direct monitoring of zinc oxidation current in the absence and in the presence of the oligo. Zinc titration curves with the ds-oligodeoxyribonucleotide were obtained in concentrations ranging from 3.62×10 −9 to 3.62×10 −8 M and 4.06×10 −10 to 5.25×10 −9 M. The acquired data were used to determine the dissociation constant, stoichiometry and zinc binding sites of the complex and to understand the specific changes of ds-oligodeoxyribonucleotide secondary structure by zinc binding. The oxidation–reduction process of zinc was also investigated by cyclic voltammetry through I (oxidation current) versus v 1/2 (square root of scan rate) curves in the absence and in the presence of the double-stranded oligodeoxyribonucleotide.

Oxidative DNA Damage Associated with Combination of Guanine and Superoxide Radicals and Repair Mechanisms via Radical Trapping

In living tissues under inflammatory conditions, superoxide radicals (O(2)*)) are generated and are known to cause oxidative DNA damage. However, the mechanisms of action are poorly understood. It is shown here that the combination of O(2)* with guanine neutral radicals, G(-H)* in single- or double-stranded oligodeoxyribonucleotides (rate constant of 4.7 +/- 1.0 x 10(8) m(-1) s(-1) in both cases), culminates in the formation of oxidatively modified guanine bases (major product, imidazolone; minor product, 8-oxo-7,8-dihydroguanine). The G(-H)* and O(2)* radicals were generated by intense 308 nm excimer laser pulses resulting in the one-electron oxidation and deprotonation of guanine in the 5'-d(CC[2AP]-TCGCTACC) strands and the trapping of the ejected electrons by molecular oxygen (Shafirovich, V., Dourandin, A., Huang, W., Luneva, N. P., and Geacintov, N. E. (2000) Phys. Chem. Chem. Phys. 2, 4399-4408). The addition of Cu,Zn-superoxide dismutase, known to react rapidly with superoxide, dramatically enhances the life-times of guanine radicals from 4 to 7 ms to 0.2-0.6 s in the presence of 5 microm superoxide dismutase. Oxygen-18 isotope labeling experiments reveal two pathways of 8-oxo-7,8-dihydroguanine formation including either addition of O(2)* to the C-8 position of G(-H)* (in the presence of oxygen), or the hydration of G(-H)* (in the absence of oxygen). The formation of the guanine lesions via combination of guanine and superoxide radicals is greatly reduced in the presence of typical antioxidants such as trolox and catechol that rapidly regenerate guanine by the reductive "repair" of G(-H)* radicals. The mechanistic aspects of the radical reactions that either regenerate undamaged guanine in DNA or lead to oxidatively modified guanine bases are discussed.

Early transcriptional activation of the hsp70.1 gene by osmotic stress in one-cell embryos of the mouse.

In fertilized mouse eggs, de novo transcription of embryonic genes is first observed during the S phase of the one-cell stage. This transcription, however, is mostly limited to the male pronucleus and possibly uncoupled from translation, making the functional meaning obscure. We found that one-cell mouse embryos respond to the osmotic shock of in vitro isolation with migration of HSF1, the canonical stress activator of mammalian heat shock genes, to pronuclei and by transient transcription of the hsp70.1, but not hsp70.3 and hsp90, heat shock genes. Isolated growing dictyate oocytes also display a nuclear HSF1 localization, but, in contrast with embryos, they transcribe both hsp70.1 and hsp70.3 genes only after heat shock. Intranuclear injection of double-stranded oligodeoxyribonucleotides containing HSE, GAGA box or GC box consensus sequences, and antibodies raised to transcription factors HSF1, HSF2, Drosophila melanogaster GAGA factor, or Sp1 demonstrated that hsp70.1 transcription depends on HSF1 in both oocytes and embryos and that Sp1 is dispensable in oocytes and inhibitory in the embryos. Hsp70.1 thus represents the first endogenous gene so far identified to be physiologically activated and tightly regulated after fertilization in mammals.

Specific conjugation of DNA binding proteins to DNA templates through thiol–disulfide exchange

The double-stranded oligodeoxyribonucleotides with single internucleotide disulfide linkages were successfully used for covalent trapping of cysteine containing protein. In particular, an efficient conjugation of DNA methyltransferase SsoII to sequence-specific decoys was demonstrated. The obtained results assume that synthetic oligodeoxyribonucleotides bearing a new trapping site can be used as new tools to study and manipulate biological systems.

Abetimus: Abetimus sodium, LJP 394.

Abetimus [Abetimus sodium, LJP 394, Rentol, Riquent] is a synthetic Toleragen molecule consisting of four double-stranded oligodeoxyribonucleotides attached to nonimmunogenic polyethylene glycol, a proprietary carrier platform. It was originated by La Jolla Pharmaceuticals. Abetimus is an immunomodulating agent that induces tolerance in B cells directed against double-stranded DNA (dsDNA). It does this by cross-linking surface antibodies. These antibodies are thought to be responsible for lupus nephritis, a chronic kidney disease that develops in patients with systemic lupus erythematosus. A phase III trial of abetimus was completed in December 2002. La Jolla Pharmaceuticals previously established two licensing agreements for abetimus, which have since been terminated. One of the agreements was with Leo Pharmaceutical Products of Denmark. The company was licensed rights to abetimus covering Europe and the Middle East. The other agreement was with Abbott Laboratories. Abbott returned all rights to abetimus to La Jolla Pharmaceuticals in September 1999 based on the results of an analysis of the phase II/III trial of abetimus in lupus patients with a history of renal disease. A phase III trial, named "PEARL" (Program Enabling Antibody Reduction in Lupus), has been conducted in the US in patients with lupus nephritis. It enrolled 317 patients with a history of lupus who were treated with a weekly dose of abetimus 100mg or placebo. The trial was completed in December 2002 and preliminary results were reported in February 2003. PEARL was designed to determine whether abetimus can significantly delay renal flares and delay the need for treatment with high-dose corticosteroids and/or cyclophosphamide in patients with high affinity IgG antibodies to the double-stranded oligonucleotide epitope in abetimus. Patients with high-affinity antibodies, were selected using a surface plasmon resonance (SPR)-based pharmacoproteonomics assay provided by Biacor International. Following the completion of PEARL in December 2002, La Jolla Pharmaceuticals initiated an on-going, open-label, follow-on trial. All patients who had completed PEARL were eligible to enroll and receive weekly treatment with abetimus. However, in April 2003, La Jolla Pharmaceuticals announced that it was closing this trial, which was designed to collect additional long-term safety data, to conserve resources for the continued development of the drug. Previously, La Jolla Pharmaceuticals and Abbott initiated a phase II/III trial of abetimus in more than 200 patients with lupus nephritis. However, this trial was discontinued in May 1999 because the primary end-point (time to renal flare) was much shorter than expected. After the trial was halted, further analysis of trial data using a new blood test to measure the strength of the binding between abetimus and a patient's antibodies to dsDNA was conducted. The additional analysis showed that the number of renal flares in the high-affinity patients (responders) treated with abetimus was less than half of the number of renal flares in high-affinity patients treated with placebo. Responders also showed a significant reduction in the use of high-dose corticosteroids and cyclophosphamide. Being able to screen patients to identify those likely to respond to therapy lead La Jolla Pharmaceuticals to initiate PEARL after Abbott's withdrawal from the drug. It is believed that screening patients will help increase the cost-effectiveness of clinical development. In September 2000, the US FDA granted orphan drug status for abetimus in the treatment of lupus nephritis. The European Commission followed suit in November 2001, granting orphan drug status for abetimus in the EU.

Specificity of mammalian Y-box binding protein p50 in interaction with ss and ds DNA analyzed with generic oligonucleotide microchip.

p50 protein is a member of the Y-box binding transcription factor family and is a counterpart of YB-1 protein. The generic microchip was used to analyze the sequence specificity of p50 binding to single (ss) and double-stranded (ds) oligodeoxyribonucleotides. The generic microchip contained 4,096 single-stranded octadeoxyribonucleotides in which all possible core 6-mers (4(6)=4,096) were flanked at their 3' and 5'-ends with degenerated nucleotides. The oligonucleotides were chemically immobilized within polyacrylamide gel pads fixed on a glass slide. The binding of p50 to the generic microchip was shown to be the most specific to ss GGGG motif and then to ss CACC and CATC motifs. GC-rich ds oligonucleotides of the generic microchip, and particularly those containing GGTG/CACC, GATG/CATC, and GTGG/CCAC heterogeneous motifs, were most efficiently destabilized due to interaction with p50. Gel-shift electrophoresis has shown that the protein exhibits much higher binding specificity to 24-mer oligoA-TGGGGG-oligoA containing G-rich 6-mer, in comparison with 24-mer oligoA-AAATAT-oligoA carrying A,T-rich 6-mer in full correspondence with the data obtained with the microchip. Studies of DNA-binding proteins using gel-immobilized ss and ds DNA fragments provide a unique possibility to detect low-affinity complexes of these proteins with short sequence motifs and assess the role of these motifs in sequence-specific interactions with long recognition sites.

L-methionine inhibits reaction of DNA with anticancer cis-diamminedichloroplatinum(II).

Sufficient evidence has accumulated to identify DNA as the relevant pharmacological target of antitumor cisplatin [cis-diamminedichloroplatinum(II)]. This drug is administered intravenously so that before it reaches DNA in the nucleus of tumor cells it may interact with various compounds including sulfur-containing molecules such as L-methionine or the compounds containing these residues. L-Methionine increases the rate of reaction of cisplatin with monomeric guanosine 5'-monophosphate, and it was suggested on the basis of these results previously obtained by other authors that methionine residues could mediate the transfer of platinum onto DNA. We studied in the present work the reactions of the 1:1 complex formed between cisplatin and L-methionine or N-acetyl-L-methionine with synthetic, single- and double-stranded oligodeoxyribonucleotides and natural, high molecular mass DNA by using high-pressure liquid chromatography and flameless atomic absorption spectrophotometry. The results demonstrate that both L-methionine and N-acetyl-L-methionine decrease the rate of reaction of cisplatin with base residues in natural, high molecular mass DNA. Thus, the possibility that cisplatin bound to methionine residues serves as a drug reservoir available for platination of DNA in the nucleus of tumor cells appears unlikely.