SNP detection using trityl mass tags.

A new method suitable for single nucleotide polymorphism (SNP) detection using differential oligonucleotide probe extension has been developed. Sulfur-linked laser-cleavable trityl labels are implemented in this protocol. The method is based on mass spectrometry and utilizes a single surface for affinity purification of extended probes and matrix-independent desorption-ionization of the cleavable labels.

Reactive trityl derivatives: stabilised carbocation mass-tags for life sciences applications.

The rational design of novel triarylmethyl (trityl)-based mass tags (MT) for mass-spectrometric (MS) applications is described. We propose a "pK(R+) rule" to correlate the stability of trityl carbocations with their MS performance: trityls with higher pK(R+) values ionise and desorb better. Trityl blocks were synthesised that have high pK(R+) values and are stable in conditions of MS analysis; these MTs can be ionised by matrix as well as irradiation with a 337 nm nitrogen laser.

1-, 2-, and 4-ethynylpyrenes in the structure of twisted intercalating nucleic acids: structure, thermal stability, and fluorescence relationship.

A postsynthetic, on-column Sonogashira reaction was applied on DNA molecules modified by 2- or 4-iodophenylmethylglycerol in the middle of the sequence, to give the corresponding ortho- and para-twisted intercalating nucleic acids (TINA) with 1-, 2-, and 4-ethynylpyrene residues. The convenient synthesis of 2- and 4-ethynylpyrenes started from the hydrogenolysis of pyrene that has had the sulfur removed and separation of 4,5,9,10-tetrahydropyrene and 1,2,3,6,7,8-hexahydropyrene, which were later converted to the final compounds by successive Friedel-Crafts acetylation, aromatization by 2,3-dichloro-5,6-dicyano-1,4-benzoquinone, and a Vilsmeier-Haack-Arnold transformation followed by a Bodendorf fragmentation. Significant alterations in thermal stability of parallel triplexes and antiparallel duplexes were observed upon changing the attachment of ethynylpyrenes from para to ortho in homopyrimidine TINAs.

DNA glue: 1-, 2- and 4-ethynylpyrenes in the structure of twisted intercalating nucleic acids (TINAs), DNA duplexes/triplexes and interstrand excimer formation.

Significant alterations in thermal stability of parallel DNA triplexes and antiparallel duplexes were observed upon changing the attachment of ethynylpyrenes from para to ortho in the structure of phenylmethylglycerol inserted as a bulge into DNA (TINA). Insertions of two ortho-TINAs as a pseudo-pair in the middle of the duplex resulted in formation of an excimer band at 505 nm for both 1- and 4-ethynylpyrene analogues.

Novel mass tags for single nucleotide polymorphism detection.

A new method suitable for single nucleotide polymorphism detection and other applications based on oligonucleotide probe extension has been developed. The method is based on mass spectrometry and utilizes a single surface for affinity purification of extended probes and matrix-independent desorption/ionization of the cleavable labels. A new family of sulfur-linked laser-cleavable trityl labels with vastly improved flying abilities is implemented in this study.

Pyrene-perylene as a FRET pair coupled to the N2'-functionality of 2'-amino-LNA.

Detection of nucleic acid hybridization via fluorescence resonance energy transfer (FRET) using pyren-1-ylmethyl and perylen-3-ylmethyl N2'-functionalized 2'-amino-LNA nucleosides incorporated into oligonucleotides exhibited a clear distance dependence of the FRET efficiency, ranging from below 10% when the fluorophores were approximately 40A apart to approximately 90% when the fluorophores were in close proximity.

1-Phenylethynylpyrene (1-PEPy) as refined excimer forming alternative to pyrene: case of DNA major groove excimer.

1-Phenylethynylpyrene fluorochrome was studied as meta- and para-derivatives of arabino-uridine-2'-carbamates in ss and dsDNA. 1-PEPy showed red-shifted emission and increased fluorescence quantum yield compared to pyrene. Although 1-PEPy has very short excited lifetime (<2.

Phenylethynylpyrene-labeled oligonucleotide probes for excimer fluorescence SNP analysis of 23S rRNA gene in clarithromycin-resistant Helicobacter pylori strains.

The use of phenylethynylpyrene excimer forming pair in the design of specific fluorescent probes for determination of A2144G (A2143G and/or A2143C) mutations in 23S rRNA gene of Helicobacter pylori is described. Analysis of fluorescence spectra of model duplexes revealed optimal positions of fluorophore residues in the probe sequences for maximum efficiency of SNP detection. Application of excimer forming probes for analysis of DNA samples isolated from natural bacterial strains of H.

Fluorescent 5-alkynyl-2'-deoxyuridines: high emission efficiency of a conjugated perylene nucleoside in a DNA duplex.

Four fluorescent 5-alkynyl-2'-deoxyuridines were studied in DNA oligonucleotides and their duplexes. The fluorescence response to hybridization differs dramatically for nucleosides containing a perylene fluorochrome either conjugated or not conjugated to the nucleobase. The conjugated nucleoside, 5-(perylen-3-ylethynyl)-2'-deoxyuridine, shows enhanced long-wavelength emission in the DNA duplex, in contrast to the blue fluorescence of perylene on a flexible linker (in 5-[(perylen-3-yl)methoxyprop-1-ynyl]-2'-deoxyuridine), which is quenched upon duplex formation.

5-Arylethynyl-2'-deoxyuridines, compounds active against HSV-1.

Three new 5-arylethynyl-2'-deoxyuridines containing bulky aryls have been prepared and tested against HSV-1 in Vero cells. The introduction of a substituent in the phenyl group of an inactive compound, 5-phenylethynyl-2'-deoxyuridine, leads to the appearance of anti-HSV properties. The most active compounds are those containing a polycyclic aromatic hydrocarbon residue attached to the 5 position of 2'-deoxyuridine through a rigid triple bond.

Perylene attached to DNA through stiff or flexible linker: duplex stability and FRET.

Two fluorescent nucleosides, 5-(perylen-3-ylethynyl)-2'-deoxyuridine and 5-[(perylen-3-yl)methoxypropyn-1-yl]-2'-deoxyuridine, were incorporated into synthetic oligodeoxyribonucleotides and spectral properties of the conjugates and their duplexes were studied.

Pyrenemethyl ara-uridine-2'-carbamate: a strong interstrand excimer in the major groove of a DNA duplex.

The synthesis of new nucleoside derivatives, ara-uridine-2'-carbamates, and their incorporation into synthetic DNA oligomers is described. The modification directs ligands into the major groove of duplex DNA and somewhat destabilizes the duplexes of modified oligonucleotides with complementary DNA or RNA. In the case of pyrenemethyl carbamate modification in DNA-DNA duplexes, the destabilization is considerably reduced.