Kazushige Yamana

Pyrene is highly emissive when attached to the RNA duplex but not to the DNA duplex: the structural basis of this difference

Through binding and fluorescence studies of oligonucleotides covalently attached to a pyrene group via one carbon linker at the sugar residue, we previously found that pyrene-modified RNA oligonucleotides do not emit well in the single-stranded form, yet the attached pyrene emits with a significantly high quantum yield upon binding to a complementary RNA strand. In sharp contrast, similarly modified pyrene–DNA probes exhibit very weak fluorescence both in the double-stranded and single-stranded forms. The pyrene-modified RNA oligonucleotides therefore provide a useful tool for monitoring RNA hybridization. The purpose of this paper is to present the structural basis for the different fluorescence properties of pyrene-modified RNA/RNA and pyrene-modified DNA/DNA duplexes. The results of absorption, fluorescence anisotropy and circular dichroism studies all consistently indicated that the pyrene attached to the RNA duplex is located outside of the duplex, whereas the pyrene incorporated into the DNA duplex intercalates into the double helix. 1H NMR measurements unambiguously confirmed that the pyrene attached to the DNA duplex indeed intercalates between the base pairs of the duplex. Molecular dynamics simulations support these differences in the local structural elements around the pyrene between the pyrene–RNA/RNA and the pyrene–DNA/DNA duplexes.

Bis-pyrene labeled DNA aptamer as an intelligent fluorescent biosensor

The site-directed incorporation of bis-pyrenyl fluorophore into anti-ATP DNA aptamer results in a creation of an intelligent fluorescent sensor with high signal intensity and specificity for detecting the target ligand in a homogeneous system.

Synthesis of oligonucleotide derivatives containing pyrene labeled glycerol linkers: Enhhanced excimer fluorescence on binding to a complementary DNA sequence

Abstract The synthesis of a pyrene labeled glycerol linker for multiple internal labeling of oligonucleotides has been described. The oligonucleotide containing two successively incorporated pyrenyl residues exhibits enhanced excimer fluorescence upon hybridization to the complementary DNA segment.

Synthesis of oligonucleotide derivatives with pyrene group at sugar fragment

Abstract The synthesis of oligonucleotide derivatives possessings pyrene group at a specific sugar residue has been described. The incorporation of the pyrene was accomplished by preparation of the pyrene-modified uridine, 2′-(1-pyrenylmethyl)uridine, which was then converted to the protected phosphorobisdiethylamidite. This reagent was used for the solid-phase synthesis of oligonucleotide-pyrene conjugates. The purification of the conjugates was effected by reversed-phase HPLC. The oligonucleotide-pyrene conjugate synthesized here can bind to their complementary sequences with a cooperative interaction between the pyrene group and the adjacent base-pairs.

Synthesis of a new photoisomerizable linker for connecting two oligonucleotide segments

Abstract synthesis of a photoisomerizable linker containing an azobenzene unit for connecting oligonucleotide segments has been described. 4,4′-Azobenzene dicarbonyl chloride was converted to the fully protected diol by treatment with the O-silyl protected aminopentanol. Partial deprotection of the silyl group afforded the monoprotected diol, which was protected by a dimethoxytrityl group. Then the remaining silyl protecting group was removed to afford the alcohol. The hydroxyl function was converted to the phosphoramidite. The amidite can be used for joining two oligonucleotides via the 5′-terminal hydroxyl group of one oligomer and the 3′-terminal oxygen of the other.

Enhanced Fluorescence in the Binding of Oligonucleotides With a Pyrene Group in the Sugar Fragment to Complementary Polynucleotides

Abstract The fluorescence intensity and lifetime of oligonucleotides with a pyrenylmethyl group at the specific sugar residue were increased upon binding to their complementary polynucleotide in aqueous solution. The present oligonucleotide-pyrene conjugates provide new fluorescent probes for detection of specific nucleic acids.

Synthesis, binding and fluorescence properties of oligonucleotide derivatives having a dansyl fluorescence label attached to the 2′-position of a ribonucleoside

Abstract Oligonucleotide derivatives having a dansyl fluorescence label at the 2′-position of a ribonucleoside have been synthesized by using 5′-dimethoxytrityl 2′-(dansylamino)uridine 3′-phosphorobisdiethylamidite. The oligonucleotide containing a dansyl-modified uridine at the 5′-terminal position exhibits normal binding affinity for a complementary DNA segment in an aqueous buffer solution (pH 7.0). A significant increase in fluorescence intensity and blue-shift of the emission maximum were observed for dansyl-modified oligonucleotide upon binding to DNA. The fluorescence increase upon binding of dansyl-modified oligonucleotide to the target DNA was found to be sensitive to base mismatch in the DNA sequence.

Oligonucleotides having covalently linked anthracene at specific sugar residue: Differential binding to DNA and RNA and fluorescence properties

Abstract Oligonucleolide 15 -mers containing 2-anthracenylmethyl groups at specific sugar residues have been synthesized from the 2′-anhracene-modified undine. The modified oligonucleotides bind to both the complementary DNA and RNA segments. The induced CD at around 375 nm due to the anthracene chromophore was seen only for the anthracene-modified duplexes with DNA but not for those with RNA, which is in good agreement with the modification-induced stability of the duplexes. The oligonucleotide with anthracene at the specified sugar residue exhibits enhanced fluorescence upon binding to the complementary RNA segment.

Helical pyrene-array along the outside of duplex RNA

A helical pyrene-array was formed by incorporation of multiple pyrenes into the sugar residues of duplex RNA leading to strong excimer fluorescence.

Coumarin–fluorescein pair as a new donor–acceptor set for fluorescence energy transfer study of DNA

Abstract A method for introduction of the 2′-coumarin labeled nucleoside as a fluorescence energy donor into DNA duplexes has been described. Efficient FRET occurs between the coumarin–fluorescein pair in DNA owing to the high quantum yield of the donor. The present donor–acceptor pair may be useful as FRET indicator of DNA structures in solution.