Synthesis and properties of oligonucleotides containing 2'-pyrenylalkyluridine.

The synthesis, binding and fluorescence properties of oligonucleotides containing the uridine modified at the 2'-position by a pyrene group using different length of linker arm have been described. It is demonstrated that the oligonucleotides possessing a C3-amide group at the 2'-position display an enhanced signal of the pyrene monomer fluorescence upon binding to DNA segments.     

Synthesis and properties of oligonucleotides containing a 7-membered (oxepane) sugar ring

Herein we describe the synthesis of novel 7-membered ring (oxepane) thymine and adenine nucleosides (oT and oA) and their corresponding 5'-O-phosphoramidite derivatives. Two homopolymeric sequences (oT(15) and oA(15)) were prepared via conventional solid-phase synthesis. The mutually complementary strands had the ability to form a duplex (oT(15):oA(15)) exhibiting a transition temperature of 12 degrees C. The oxepane oligonucleotides were also found to associate with their respective complementary RNA strands thus forming oT(15):rA(15) (13 degrees C) and oA(15):rU(15) (12 degrees C) hybrids. The corresponding native duplexes, namely dT(15):dA(15), dT(15):rA(15) and dA(15):rU(15) had melting temperatures of 37 degrees C, 32 degrees C and 16 degrees C, respectively. The CD spectrum of oT(15):rA(15) closely resembled that of the native dT(15):rA(15) hybrid and, in fact, both were found to be substrates for E. Coli RNase H. Thus the oxepane nucleic acids reported here are one of only a handful of DNA mimics capable of activating RNase H when bound to RNA.     

Synthesis and properties of oligonucleotides containing 8-bromo-2'-deoxyguanosine

The preparation of oligonucleotides containing 8-bromo-2'-deoxyguanosine is described. Substitution of G by 8-bromoguanine on an alternating CG decamer stabilizes the Z-form in such a way that the B-form was not observed. Melting temperatures showed that duplexes in which 8-bromo-2'-deoxyguanosine paired with natural bases were much less stable.     

Synthesis and properties of oligonucleotides containing 2'-O-methyl-2-thiouridine.

New methods to synthesize 2'-O-methyl-2-thiouridine and its phosphoramidite building block for incorporation into oligonucleotides were developed. Oligonucleotides containing 2'-O-methyl-2-thiouridine were expected to be favorable as antisense agents in several respects, i.e., nuclease resistance, stable RNA duplex formation, and exact base recognition. Therefore, to make them clear, we synthesized oligonucleotides having 2'-O-methyl-2-thiouridine and analyzed their properties in detail.     

Synthesis and properties of oligonucleotides containing 2''-deoxynebularine and 2''-deoxyxanthosine.

The preparation of synthetic oligonucleotides containing 2'-deoxynebularine (dN) and 2'-deoxyxanthosine (dX) is described. The thermal stabilities of duplexes containing dX, dN, and 2'-deoxyinosine (dI) base-paired with the four natural bases have been measured. Xanthine base pairs have stabilities at pH 5.5 that are similar to those of dI-containing duplexes at neutral pH. When xanthine is paired with adenine or cytosine an unusual stabilization of the duplex structure is observed at acid pH. Incorporation of base mispairs opposite template xanthine sites were measured using Drosophila DNA polymerase alpha. The relative nucleoside incorporation rates are in the order: T greater than C much greater than A approximately equal to G. These rates do not correlate with relative thermodynamic stabilities of base mispairs with xanthine obtained from Tm measurements: T greater than G greater than A approximately equal to C. We suggest that DNA polymerase misinsertion rates are greatest when the base mispair can be formed in accordance with Watson-Crick as opposed to other base pairing geometries even though other geometries, e.g. wobble, may result in a more stable final DNA product.     

Synthesis and hybridization properties of oligonucleotides containing (2S,3R)-9-(2,3,4-trihydroxybutyl)adenine

The synthesis and properties of oligonucleotides (ONs) containing 9-(2,3,4-trihydroxybutyl)adenine, A(C2) and A(C3), are described. The ON containing A(C2) involves the 3'-->4' and 3-->5' phosphodiester linkages in the strand, whereas that containing A(C3) possesses the 3'-->4' and 2'-->5' phosphodiester linkages. It was found that incorporation of the analogs, A(C2) or A(C3), into ONs significantly reduces the thermal and thermodynamic stabilities of the ON/DNA duplexes, but does not largely decrease the thermal and thermodynamic stabilities of the ON/RNA duplexes as compared with the case of the ON/DNA duplexes. It was revealed that the base recognition ability of A(C2) is greater than that of A(C3) in the ON/RNA duplexes.     

Synthesis and hybridization properties of oligonucleotides containing 2'-O-modified ribonucleotides.

A versatile, general way is described for the introduction of different functional groups into oligonucleotides by means of a simple linker at the 2'-position of the sugar. Nucleotide building blocks carrying lipophilic, intercalating or tertiary amino groups can be placed deliberately at any desired position of oligonucleotides by standard automated oligonucleotide synthesis. Thermal denaturation studies with these oligonucleotides reveal the following general trends: i) Modification with lipophilic n-octyl groups has little if any effect on duplex stability; a destabilizing (lipophilic) substituent is better tolerated at or near the ends than in the middle of the oligo. ii) An intercalating substituent (2-aminoanthraquinone) substantially increases duplex stability. iii) N,N-Dimethyl amino residues also increase duplex stability though to a smaller extent than intercalating residues. iv) Modifications at the 5'-end have a more pronounced influence on the TM than the corresponding 3'-modifications. v) Oligonucleotides modified in such a way show little or no loss in sequence specificity.     

Synthesis and properties of oligonucleotides containing a cholesterol thymidine monomer

Highly selective base-pair recognition makes DNA a suitable building block for orderly self-assembled structures. For some applications in nanotechnology DNA complexes need to be fixed onto surfaces. To fulfil this requirement on lipid membranes we have synthesised a thymidine monomer modified with a cholesterol moiety. Solution studies show that the melting temperature (Tm) of the duplex, with adjacent cholesterols on each strand, is much higher than that of the unmodified duplex.     

Synthesis and properties of oligonucleotides containing 4-thiothymidine, 5-methyl-2-pyrimidinone-1-beta-D(2''-deoxyriboside) and 2-thiothymidine.

Methods are given for the synthesis of derivatives of 4-thiothymidine (4ST), 5-methyl-2-pyrimidinone-1-beta-D(2'-deoxyriboside) (4HT) and 2-thiothymidine (2ST) suitable for incorporation into oligodeoxynucleotides by the cyanoethyl phosphoramidite method. 4HT and 2ST are incorporated with no base protection but the sulphur atom in 4ST is protected with an S-sulphenylmethyl (-SCH3) function. This can be removed with dithiothreitol after synthesis. These T analogues have been incorporated into GACGATATCGTC, a self-complementary dodecamer containing the Eco RV recognition site (underlined), in place of the two T residues within this site. Although pure dodecamers are obtained in each case the syntheses are not as efficient as those seen when normal unmodified bases are used mainly due to the chemical reactivity of 4ST, 4HT and 2ST. Some of the chemical properties of oligonucleotides containing these bases (reactivity towards NH3) as well as their physical properties (melting temperatures, U.V., fluorescence and circular dichroism spectra) have been determined and are discussed.     

Synthesis and triplex binding properties of oligonucleotides containing a novel nucleobase

The thiazolo-indole compound 1 bearing the complementary donor-acceptor-donor sites (dad) was designed for specific recognition of an AT inverted base pair in pyrimidine triple helix motif. It was successfully incorporated into 14-mer oligonucleotide using a serinol unit as sugar derivative. The triple helix hybridization studies were examined by means of thermal denaturation experiments with a 26-mer DNA duplex containing the AT inverted base pair.     

Synthesis and properties of oligonucleotides containing aminodeoxythymidine units.

Procedures are described for synthesis via solid support methodology of oligonucleotide analogues derived in part from 3'-amino-3'-deoxythymidine or 5'-amino-5'-deoxythymidine. Oligothymidylate decamers terminated with a 3'-amino group or containing a 3'-NHP(O)(O-)O-5' internucleoside link are found to form unusually stable complexes with poly(dA), poly(A), and oligo(dA). For related derivatives of 5'-amino-5'-deoxythymidine enhancement is less or absent, and in the case of multiple substitution destabilization of the heteroduplex may be observed. That the effect of the 3'-amino group is general for oligonucleotide derivatives is indicated by enhanced Tm values for heteroduplex complexes of the mixed-base oligomer, d(TATTCAGTCAT(NH2)), and the methyl phosphonate derivatives, TmTmTmTmTmTmTmTmTmT(NH2) and d(TmAmTmTmCmAmGmTmCmAmT(NH2)).     

Synthesis and properties of cationic 2'-O-[N-(4-aminobutyl)carbamoyl] modified oligonucleotides

2'-O-[N-(4-Aminobutylcarbamoyl)]uridine (U(abcm)) was synthesized and incorporated into oligonucleotides. The oligonucleotides incorporating U(abcm) formed more stable duplexes with their complementary and mismatched RNAs than those containing 2'-O-carbamoyluridine (U(cm)). The stability of duplex with a U(abcm)-rG base pair showed higher thermostability than the duplex having unmodified U-rG base pair. The U(abcm) residue showed enhanced resistance to snake venome phosphodiesterase.     

Synthesis and physical properties of phosphatidylcholine labelled with 9-(2-anthryl)nonanoic acid,a new fluorescent probe

Synthesis and physical properties of a new anthracene fatty acid, 9-(2-anthryl)nonanoic acid, and the corresponding anthracene-phosphatidylcholines which were obtained by condensing the acid with sn-1-palmitoyl-lysophosphatidylcholine (PAPC) and with egg lysophosphatidylcholine (EAPC) are described. Differential scanning calorimetry experiments show that these lipids can undergo a liquid-crystal to gel phase transition at temperatures of 15°C and 18°C for EAPC and PAPC, respectively. In monolayers, PAPC exhibits a compression curve nearly superimposable to that of dipalmitoylphosphatidylcholine (DPPC), with a molecular area of 0.48 nm² at π = 30 mN m^?1. The data indicate that in these lipids, the anthracene group is only slightly more bulky than a normal acyl chain and that it does not significantly affect the regular phospholipid molecular packing. In ethanol solutions or when incorporated into egg phosphatidylcholine liposomes in a molar ratio of 1%, these lipids display UV absorption spectra and fluorescence emission spectra similar to those of 2-methyl anthracene. For EAPC liposomes, a broad and structureless fluorescence emission spectrum centered at around 450 nm, was recorded, suggesting the occurrence of anthracene excimers. As ascertained by UV spectrophotometry, differential scanning calorimetry, fluorescence polarization and anthracene photodimerization experiments, EAPC displays good miscibility properties with lipids in the liquid state (egg phosphatidylcholine) or in the gel state (distearoylphosphatidylcholine (DSPC)). The potential of these anthracene derivatives for studying the dynamics and the topological distribution of lipids in biomembranes is discussed.     

Synthesis and hybridization studies of oligonucleotides containing 1-(2-deoxy-2-alpha-C-hydroxymethyl-beta-D-ribofuranosyl)thymine (2'-alpha-hm-dT)

We report the first investigation of oligoribonucleotides containing a few 1-(2-deoxy-2-alpha-C-hydroxymethyl-beta-D-ribofuranosyl)thymine units (or 2'-hm-dT, abbreviated in this work as 'H'). Both the 2'-CH2O-phosphoramidite and 3'-O-phosphoramidite derivatives of H were synthesized and incorporated into both 2',5'-RNA and RNA chains. The hybridization properties of the modified oligonucleotides have been studied via thermal denaturation and circular dichroism studies. While 3',5'-linked H was shown previously to significantly destabilize DNA:RNA hybrids and DNA:DNA duplexes (modification in the DNA strand; DeltaT(m) approximately -3 degrees C/insert), we find that 2',5'-linked H have a smaller effect on 2',5'-RNA:RNA and RNA:RNA duplexes (DeltaT(m) = -0.3 degrees C and -1.2 degrees C, respectively). The incorporation of 3',5'-linked H into 2',5'-RNA:RNA and RNA:RNA duplexes was found to be more destabilizing (-0.7 degrees C and -3.6 degrees C, respectively). Significantly, however, the 2',5'-linked H units confer marked stability to RNA hairpins when they are incorporated into a 2',5'-linked tetraloop structure (DeltaT(m) = +1.5 degrees C/insert). These results are rationalized in terms of the compact and extended conformations of nucleotides.     

Synthesis of oligonucleotides containing a new azobenzene fragment with efficient photoisomerizability

The azobenzene derivatives possessing substituents of ROCH₂CH₂O- and-CH₂CH₂OR′ or -CONHCH₂CH₂OR′ at p,p′-positions, where R and R′ are 4,4′-dimethoxytrityl and 2-cyanoethyl-N,N′-diisopropylphophoramidite, have been synthesized for linking two oligonucelotide segments. It has been found that the azobenzene linkers efficiently undergo trans–cis isomerization by exposing to UV light. The conversion efficiency showed slight dependence on structure or conformation of oligonucleotides attached to the azobenzene chromophore. The cis-form of the azobenzene in oligonucleotides was sufficiently stable at low temperature under dark. The present findings would open the way for light switch of nucleic acid structures.     

Synthesis and properties of DNA-duplexes, containing 9-(1'-hydroxy-2'-(hydroxymethyl)ethoxy)methylguanine]

Phosphoramidite derivative of 9-[1'-hydroxy-2'-(hydroxymethyl)ethoxy]methylguanine (glG) is synthesized which allows one to introduce point modifications in any position of the chemically prepared oligonucleotide chain. Oligonucleotides with 5'-terminal glG can be used in chemical ligation promoted by cyanogen bromide. The modified oligonucleotide duplexes were characterized by melting curves and CD spectra.     

Synthesis and Fluorescent Characteristics of Oligodeoxynucleotides Containing a Novel Fluorescent Label,p-(2-Benzoxazolyl)tolane

A functional (dihydroxybutyl) derivative of p-(2-benzoxazolyl)tolane, a fluorescent label novel for biopolymers, was synthesized. The functionalized solid support obtained on its basis was employed in the synthesis of oligodeoxynucleotides 3-terminally labeled with benzoxazolyltolane (these oligonucleotides also contained 1-phenylethynylpyrene residues). This fluorophore within its dihydroxybutyl derivative and the oligonucleotides modified with it display an intensive fluorescence characterized by a high Stokes shift. The oligonucleotides labeled with this fluorophore are potential probes sensitive to the bioplymer microenvironment.     

Synthesis of DNA oligonucleotides containing 5-(mercaptomethyl)-2'-deoxyuridine moieties

Recently thiolated oligonucleotides have attracted significant interest due to their ability to efficiently undergo stable bond formation with gold nanoparticles and surfaces to form DNA conjugates. In this respect we became interested in the synthesis of oligonucleotides that bear short thioalkyl functions located at the nucleobase. Here we present a strategy for the synthesis of DNA oligonucleotides that bear 5-(mercaptomethyl)-2'-deoxyuridine moieties. The building blocks were synthesized in a straightforward manner from thymidine. Only moderate changes of standard protocols for automated DNA synthesis are required for the generation of modified oligonucleotides containing the thiolated building blocks.     

Synthesis and hybridization properties of oligonucleotides containing 6-membered azasugar nucleotides

A modified nucleoside was synthesized with adenine and a 6-membered azasugar, and it was converted to the phosphoramidite which was used for the incorporation into oligonucleotides. The hybridization properties of the modified oligonucleotides with DNA and RNA were studied.