G. A. Maksakova

Universal oligonucleotide microarray for sub-typing of Influenza A virus.

A universal microchip was developed for genotyping Influenza A viruses. It contains two sets of oligonucleotide probes allowing viruses to be classified by the subtypes of hemagglutinin (H1–H13, H15, H16) and neuraminidase (N1–N9). Additional sets of probes are used to detect H1N1 swine influenza viruses. Selection of probes was done in two steps. Initially, amino acid sequences specific to each subtype were identified, and then the most specific and representative oligonucleotide probes were selected. Overall, between 19 and 24 probes were used to identify each subtype of hemagglutinin (HA) and neuraminidase (NA). Genotyping included preparation of fluorescently labeled PCR amplicons of influenza virus cDNA and their hybridization to microarrays of specific oligonucleotide probes. Out of 40 samples tested, 36 unambiguously identified HA and NA subtypes of Influenza A virus.

Interaction of Endonuclease Eco RI with Short Specific and Nonspecific Oligonucleotides

The interaction of Eco RI with different oligodeoxyribonucleotides (ODNs) was analyzed using the method of the slow step‐by‐step simplification in their complexity. Orthophosphate (K I = 31 mM), 2‐deoxyribose 5‐phosphate (K I = 4.6 mM) and different dNMPs (K I = 2.1‐2.5 mM) were shown to be the minimal ligands of the enzyme. The lengthening of a nonspecific d(pN) n (n = 1‐6) by one nucleotide unit resulted in the increase of their affinity by a factor of 2.0. Weak nonspecific electrostatic contacts of Eco RI with internucleotide phosphate groups of ODNs can account for about 5 orders of magnitude in the ligand affinity, whereas the contribution of specific interactions between Eco RI and d(pN) n is no more than 2 orders of magnitude of a total ODNs affinity.

The influence of the target structure on the efficiency of alkylation of single‐stranded DNA with the reactive derivatives of antisense oligonucleotides

Site‐directed alkylation of three oligonucleotide targets: 41‐mer (hairpin structure), 22‐mer (loop part of this hairpin) and 10‐mer (part of the loop) with 5′‐p‐(N‐2‐chloroethyl‐N‐methylamino)benzylamides of oligonucleotides complementary to the loop region was studied. Thermodynamic parameters of the interaction were estimated using the dependence of the limit modification extent on the reagent concentration at several temperatures. The stability of the complex increases significantly in the set: 302‐mer carrying above hairpin, 41‐mer, 22‐mer, the data for 22‐mer and 10‐mer being nearly identical. This indicates significant influence of the loop supporting structure on the interaction with antisense reagents.

Intraduplex Photo-cross-linking of p-Azidoaniline Residue and Amino Acid Side Chains Linked to the Complementary Oligonucleotides via a New Phosphorylating Intermediate Formed in the Mukaiyama System

Abstract Oligonucleotide derivatives carrying a side chain of either lysine or histidine at the 3′-end and their complementary oligonucleotides having photoreactive groups a p-azidophenyl-NH(CH2)nNH- (n = 4, 6) residue at the 5′-end were prepared by using new phosphorylating species formed by treatment of oligonucleotides with Ph3P and (PyS)2 or (PyrS)2. in DMF, DMSO or their mixture. Efficient cross-linking of duplexes occurred under UV-irradiation (λ > 300 nm).

The algorithm of estimation of the Km values for primers in DNA synthesis catalyzed by human DNA polymerase α

DNA synthesis with various deoxyribo homo‐ and heterooligoprimers in the presence of complementary templates was investigated. The lengthening of d(pN) n primers (n =1–10) by a unit resulted in an increase of the primer affinity and a maximal rate of polymerization. The coefficient of the affinity enhancement of primers due to formation of one hydrogen bond between primer and template was found to be 1.35. The dependence of the primer affinity and polymerization rate on template‐primer structure in solution was analyzed and the objective laws of the changes of the KM and V max values were revealed.

TaqMan probes based on oligonucleotide-hairpin minor groove binder conjugates

Hybridization of TaqMan probes derived from oligonucleotides containing fluorophores (fluorescein, FAM, or tetramethylrhodamine, (Tamra)), fluorescence quenchers (BHQ1 or BHQ2), and a conjugated hairpin binder (MGB) composed of two tripyrrolcarboxamide residues connected through an aminobutyric acid residue were proposed for discrimination of single base mismatch using the real time PCR technique. Identification of A/C mismatch was shown to be highly specific for hepatitis C virus subtypes 1a and 1b with two variants of the probe (5′-3′): Tamra-ATTGAGCGGGTTTAp-BHQ2-MGB for subtype 1a and FAMATTGAGCGGGTTGAp-BHQ1-MGB for subtype 1b. Perfect duplexes (A·T-and G·C pairs) increase fluorescence in the process of amplification, whereas imperfect duplexes (A·G-and T·C pairs) induce no fluorescence changes. This phenomenon enables simultaneous genotyping of hepatitis C virus subtypes 1a and 1b.

Subtyping of influenza virus A hemagglutinin with a hybridization microarray

An oligonucleotide microarray for influenza A hemagglutinin subtyping was presented. The number of probes for the determination of each subtype of hemagglutinin (H1-H13, H15, H16, pandemic flu H1N1) varied from 13 to 28. When testing the microarray using 40 type-A influenza virus isolates, the hemagglutinin subtypes were unambiguously determined for 36 specimens.

Oligonucleotide microarray for the subtyping of influenza virus a neuraminidase

A microarray for the subtyping of influenza A neuraminidase is presented. The selection of oligoprobes proceeded in two steps. The first step included the selection of peptides specific for each subtype of neuraminidase. At the second step, the oligoprobes were calculated using the found peptide structures with the subsequent additional selection of the most specific and representative probes. From 19 to 24 probes were used for the determination of each neuraminidase subtype. The microarray testing for 19 samples with the most widespread types (N1 and N2) specifies in an unequivocal definition 18 of them and only 1 isolate has not been identified.

A second generation universal microarray for subtyping influenza a virus

The microchip for influenza A subtyping working on the “one spot-one subtype” principle was developed. Each spot contains a set of oligonucleotide probes specific for particular subtypes of hemagglutinin, neuraminidase and matrix protein (influenza A marker). Reliability of the proposed chip is the same as for the full-size microchip for separate hemagglutinin and neuraminidase typing which was created in our group earlier. The image was visualized by labeling the analyzed nucleic acid by either fluorescent dye or biotin with the following fixation in streptavidin-gold nanoparticles and development by silver precipitation. In the second case, the image was analyzed using an ordinary scanner that essentially simplifies influenza A subtyping.

Sulfonium derivatives of thioxanthenone, a new class of photodetritylating agents for microarray oligonucleotide synthesis

The usability of a new class of photo acids, namely, sulfonium hexaphosphates based on thioxanthenone, for the removal of the dimethoxytrityl protective group in the process of oligonucleotide synthesis has been studied in order to search for new detritylating agents for microarray oligodeoxyribonucleotide synthesis. 2,4-Diethyl-9-oxo-10-(4-heptyloxyphenyl)-9H-thioxanthenium hexafluorophosphate has been successfully used for the solid-phase synthesis of (dT)10.