Maksim V. Kvach

Two-dye and one- or two-quencher DNA probes for real-time PCR assay: synthesis and comparison with a TaqMan™ probe.

AbstractA typical TaqMan™ real-time PCR probe contains a 5′-fluorescent dye and a 3′-quencher. In the course of the amplification, the probe is degraded starting from the 5′-end, thus releasing fluorescent dye. Some fluorophores (including fluorescein) are known to be prone to self-quenching when located near each other. This work is aimed at studying dye–dye and dye–quencher interactions in multiply modified DNA probes. Twenty-one fluorogenic probes containing one and two fluoresceins (FAM), or a FAM–JOE pair, and one or two BHQ1 quenchers were synthesized using non-nucleoside reagents and “click chemistry” post-modification on solid phase and in solution. The probes were tested in real-time PCR using an ~300-bp-long natural DNA fragment as a template. The structural prerequisites for lowering the probe background fluorescence and increasing the end-plateau fluorescence intensity were evaluated and discussed. FigureFluorogenic TaqMan probes with various modifications for real-time PCR

Azide phosphoramidite in direct synthesis of azide-modified oligonucleotides.

Azide and phosphoramidite functions were found to be compatible within one molecule and stable for months in solution kept frozen at -20 °C. An azide-carrying phosphoramidite was used for direct introduction of multiple azide modifications into synthetic oligonucleotides. A series of azide-containing oligonucleotides were modified further using click reactions with alkynes.

Determination of Concentration of Amphiphilic Polymer Molecules on the Surface of Encapsulated Semiconductor Nanocrystals

We present a method for the determination of the average number of polymer molecules on the surface of A(II)B(VI) luminescent core-shell nanocrystals (CdSe/ZnS, ZnSe/ZnS quantum dots, and CdS/ZnS nanorods) encapsulated with amphiphilic polymer. Poly(maleic anhydride-alt-1-tetradecene) (PMAT) was quantitatively labeled with amino-derivative of fluorescein and the average amount of PMAT molecules per single nanocrystal was determined using optical absorption of the dye in the visible spectral range. The average amount of PMAT molecules grows linearly with the surface area of all studied nanocrystals. However, the surface density of the monomer units increases nonlinearly with the surface area, because of the increased competition between PMAT molecules for Zn-hexanethiol surface binding sites. The average value of zeta potential (ζ = -35 mV) was found to be independent of the size, shape, and chemical composition of nanocrystals at fixed buffer parameters (carbonate-bicarbonate buffer, pH 9.5 and 5 mM ionic strength). This finding is expected to be useful for the determination of the surface density of remaining carboxyl groups in PMAT-encapsulated nanocrystals.

Simple reagent for the synthesis of oligonucleotides labeled with 3,3,3′,3′-tetramethyl-2,2′-indodicarbocyanine

Synthesis of a phosphoramidite reagent for the preparation of oligonucleotides labeled at the 5′-end with a fluorescent dye, 3,3,3′,3′-tetramethyl-2,2′-indodicarbocyanine, is described. The efficiency of this reagent is confirmed by the synthesis of several labeled oligonucleotides.

Non-nucleoside phosphoramidites of xanthene dyes (FAM, JOE, and TAMRA) for oligonucleotide labeling.

This unit describes the preparation of 5‐ and 6‐carboxy derivatives of the xanthene fluorescent dyes fluorescein (FAM), 4′,5′‐dichloro‐2′,7′‐dimethoxy‐fluorescein (JOE), and tetramethylrhodamine (TAMRA) as individual isomers, and their conversion to non‐nucleoside phosphoramidite reagents suitable for oligonucleotide labeling. The use of a cyclohexylcarbonyl (Chc) protecting group for blocking of phenolic hydroxyls facilitates the chromatographic separation of isomers of carboxy‐FAM and carboxy‐JOE as pentafluorophenyl esters. Acylation of 3‐dimethylaminophenol with 1,2,4‐benzenetricarboxylic anhydride gave a mixture of 4‐dimethylamino‐2‐hydroxy‐2′,4′(5′)‐dicarboxybenzophenones, easily separable into individual compounds upon fractional crystallization. Individual isomeric benzophenones are precursors of 5‐ or 6‐carboxytetramethylrhodamines. The dyes were converted into 6‐aminohexanol‐ (JOE), 4‐trans‐aminocyclohexanol‐ (FAM and JOE), and hydroxyprolinol‐based (TAMRA) phosphoramidite reagents. Curr. Protoc. Nucleic Acid Chem. 52:4.55.1‐4.55.33.

Reagents For The Selective Immobilization Of Oligonucleotides On Solid Supports

New reagents (CPGs and phosphoramidites) for automatic solid phase synthesis of modified oligonucleotides were designed. Three oligonucleotides carrying fluorescent label at the 5′-terminus and an anchor group at the 3′-terminus were prepared and their immobilization in orthogonal conditions on solid supports was studied.

Phosphoramidite reagents and solid-phase supports based on hydroxyprolinol for the synthesis of modified oligonucleotides

The synthesis of phosphoramidite reagents and solid-phase supports based on hydroxyprolinol for the introduction of the residues of biotin, lipoic acid, amino groups, and terminal acetylene groups at different positions of the oligonucleotide chain has been described. The efficiency of the reagents and supports has been confirmed by the synthesis of the corresponding modified oligonucleotides.

Nanoparticles-based fluorescent conjugates for MRI contrast agents and bioimaging

Magnetic resonance imaging (MRI) is a useful tool in clinical diagnostic field because it has higher spatial resolution and contrast in soft tissue than other imaging procedures. The MRI method is based on the magnetic property of protons that align themselves in a very large magnetic field and the main MRI parameters are spin-lattice (T1) and spin-spin (T2) relaxation rates and times. The contrast agents are used to shorten relaxation times and can be classified in several ways based on their chemical nature, magnetic properties, biodistribution etc. The majority of MRI contrast agents, including the most widely used gadolinium-based ones, modify T1 relaxation. Although the toxicity of many gadolinium-based contrast agents is still under discussion. The superparamagnetic Fe3O4-nanoparticles, being much less popular and acting upon T2 relaxation, are getting growing interest as they are less toxic and capable for conjugation, which gives way to new promising multimodal agents for bioimaging.