Oligonucleotide analogues bearing an acyclonucleoside linked by an internucleotide amide bond

Oligonucleotide analogues bearing an acyclocytidine linked to thymidine by an amide (3'-O-CH2-CO-N-5') bond were synthesized. Melting curves of duplexes formed by modified oligonucleotides and complementary natural oligomers were obtained and thermodynamic parameters of their formation were measured. Replacement of dCpT by a modified dinucleotide only moderately decreased the melting temperature of these modified duplexes in comparison with unmodified duplexes containing complementary natural bases. CD spectra of modified duplexes were studied, and the duplex spatial structures are discussed. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2008, vol. 34, no. 2; see also http://www.maik.ru.     

Oligonucleotide analogues containing the internucleotide linker C3′-NH-CO-CH<Subscript>2</Subscript>-N(CH<Subscript>3</Subscript>)-C5′

Oligonucleotide analogues were synthesized whose internucleoside linker contains an amide bond and a methylamino group (C3′-NH-CO-CH₂-N(CH₃)-C5′). Melting curves for duplexes formed by modified oligonucleotides and natural oligonucleotides complementary to them were measured, and the melting temperatures and thermodynamic parameters of duplex formation were calculated. The introduction of one modified dinucleoside linker into the oligonucleotide only slightly decreases the melting temperatures of these duplexes compared with unmodified ones. The CD spectra of modified duplexes were studied, and their spatial structures are discussed.     

Oligonucleotide analogues containing the internucleotide linker C3'-NH-CO-CH2-N(CH3)-C5'

Oligonucleotide analogues were synthesized whose internucleoside linker contains an amide bond and a methylamino group (C3'-NH-CO-CH2-N(CH3)-C5'). Melting curves for duplexes formed by modified oligonucleotides and natural oligonucleotides complementary to them were measured, and the melting temperatures and thermodynamic parameters of duplex formation were calculated. The introduction of one modified dinucleoside linker into the oligonucleotide only slightly decreases the melting temperatures of these duplexes compared with unmodified ones. The CD spectra of modified duplexes were studied, and their spatial structures are discussed.     

Synthesis of Photo-Responsive Acridine-Modified DNA and Its Application to Site-Selective RNA Scission

Photo-responsive phosphoramidite monomers, which bear an azobenzene between acridine and the phosphoramidite unit, were synthesized, and incorporated into oligonucleotides. Upon UV irradiation, the azobenzene in the modified DNA efficiently isomerized from the trans isomer into the cis isomer. Although the T_m values of their duplexes with complementary DNA were not much changed by the isomerization, site-selective RNA scission was significantly accelerated by the UV irradiation when Mn(II) ion was used as the catalyst for RNA scission.     

SYNTHESIS AND HYBRIDIZATION PROPERTIES OF RNA CONTAINING 8-CHLOROADENOSINE

ABSTRACT

8-Chloroadenosine (8-Cl-Ado) has shown potential as a chemotherapeutic agent for the treatment of multiple myeloma and certain leukemias. 8-Cl-Ado treatment leads to a decrease in global RNA levels and incorporation of the analog into cellular RNA in malignant cells. To investigate the effects of 8-Cl-Ado modifications on RNA structure and function, an 8-Cl-Ado phosphoramidite and controlled-pore glass support were synthesized and used to introduce 8-Cl-Ado at internal and 3′- terminal positions, respectively. RNA oligonucleotides containing 8-chloroadenine (8-Cl-A) residues were synthesized and hybridized with complementary RNA strands. Circular dichroism spectroscopy of the resulting RNA duplexes revealed that the modified nucleobase does not perturb the overall A-form helix geometry. The thermal stabilities of 8-Cl-Ado modified duplexes were determined by UV thermal denaturation analysis and were compared with analogous natural duplexes containing standard and mismatched base pairs. The 8-Cl-Ado modification destabilizes RNA duplexes by ～5 kcal/mole, approximately as much as a U:U mismatched base pair. The duplex destabilization of 8-Cl-A may result from perturbation of Watson-Crick base pairing induced by conformational preferences of 8-halogenated nucleosides.     

Oligonucleotide analogues containing a C3′-NH-C(O)-CH<Subscript>2</Subscript>-C5′ amide internucleotide bond

A dinucleotide containing a C3′-NH-C(O)-CH₂-C5′ amide internucleotide bond was synthesized by the interaction of 3′-deoxy-3′-amino-5′-O-(tert-butyldimethylsilyl)thymidine with 3′-O-benzyl-2′-O-tert-butyldimethylsilyl-5′-deoxy-5′-carboxymethylribosylthymine, which was obtained from 2′-O-acetyl-3′-O-benzyl-5′-deoxy-5′-ethoxycarbonylmethylribosylthymine through the methanolysis of the acetyl group followed by silylation of liberated hydroxyl and ester saponification. After standard manipulation with protecting groups, the dinucleotide was converted into 3′-O(2-cyanoethyl-N,N-diisopropylphosphoramidite), which was used for the synthesis of modified oligonucleotides on an automated synthesizer. The melting curves of the duplexes formed by modified and complementary natural oligonucleotides were registered, and the melting temperatures and thermodynamic parameters of the duplex formation were calculated. The introduction of a single modified bond into the oligonucleotide led to an insignificant decrease in the melting temperature of these duplexes as compared to unmodified ones.     

Synthesis and hybridization properties of RNA containing 8-chloroadenosine

8-Chloroadenosine (8-Cl-Ado) has shown potential as a chemotherapeutic agent for the treatment of multiple myeloma and certain leukemias. 8-Cl-Ado treatment leads to a decrease in global RNA levels and incorporation of the analog into cellular RNA in malignant cells. To investigate the effects of 8-Cl-Ado modifications on RNA structure and function, an 8-Cl-Ado phosphoramidite and controlled-pore glass support were synthesized and used to introduce 8-Cl-Ado at internal and 3'- terminal positions, respectively. RNA oligonucleotides containing 8-chloroadenine (8-Cl-A) residues were synthesized and hybridized with complementary RNA strands. Circular dichroism spectroscopy of the resulting RNA duplexes revealed that the modified nucleobase does not perturb the overall A-form helix geometry. The thermal stabilities of 8-Cl-Ado modified duplexes were determined by UV thermal denaturation analysis and were compared with analogous natural duplexes containing standard and mismatched base pairs. The 8-Cl-Ado modification destabilizes RNA duplexes by approximately 5 kcal/mole, approximately as much as a U:U mismatched base pair. The duplex destabilization of 8-Cl-A may result from perturbation of Watson-Crick base pairing induced by conformational preferences of 8-halogenated nucleosides.     

Synthesis and properties of oligodeoxynucleotides containing 5-carboxy-2'-deoxycytidines

5-Carboxy-2'-deoxycytidine (dC(COO-)) was synthesized as an anion-carrier to seek a new possibility of modified oligodeoxynucleotides capable of stabilization of duplexes and triplexes. The base pairing properties of this compound were evaluated by use of ab initio calculations. These calculations suggest that the Hoogsteen-type base pair of dC(COO-)-G is less stable than that of the canonical C+-G pair and the Watson-Crick-type base pair of dC(COO-)-G is slightly more stable than the natural G-C base pair. The modified cytosine base showed a basicity similar to that of cytosine (pKa 4.2). It turned out that oligodeoxynucleotides 13mer and 14mer incorporating dC(COO-) could form duplexes with the complementary DNA oligomer, which were more stable than the unmodified duplex. In contrast, it formed a relatively unstable triplex with the target ds DNA.     

C3′-endo-puckered pyrrolidine containing PNA has favorable geometry for RNA binding: Novel ethano locked PNA (ethano-PNA)

A novel peptide nucleic acid (PNA) analogue is designed with a constraint in the aminoethyl segment of the aegPNA backbone so that the dihedral angle β is restricted within 60–80°, compatible to form PNA:RNA duplexes. The designed monomer is further functionalized with positively charged amino-/guanidino-groups. The appropriately protected monomers were synthesized and incorporated into aegPNA oligomers at predetermined positions and their binding abilities with cDNA and RNA were investigated. A single incorporation of the modified PNA monomer into a 12-mer PNA sequence resulted in stronger binding with complementary RNA over cDNA. No significant changes in the CD signatures of the derived duplexes of modified PNA with complementary RNA were observed.     

Effects of 5-(N-aminohexyl)carbamoyl-2'-deoxyuridine on endonuclease stability and the ability of oligodeoxynucleotide to activate RNase H.

To evaluate an endonuclease resistance property of oligodeoxynucleotides (ODNs) containing 5-(N-aminohexyl)carbamoyl-2'-deoxyuridines (Hs) and to elucidate whether a duplex consisting of the ODN analogue and its complementary RNA induces RNase H activity, the ODNs containing the deoxyuridine analogues, Hs, at intervals of one, two, three, four and five natural nucleosides were synthesized. From partial hydrolysis of these ODNs with nuclease S1 (an endonuclease), it was found that the ODNs became more stable towards nucleolytic hydrolysis by the enzyme as the number of H increased. Furthermore, to examine whether the duplexes composed of the ODNs containing Hs and their complementary RNAs are substrates for RNase H or not, the duplexes of these ODNs and their complementary RNA strands were treated with Escherichia coliRNase H. It was found that cleavage of the RNA strands by the enzyme was kinetically affected by the introduction of Hs into the duplexes.     

THE EFFECT OF A SINGLE BORANOPHOSPHATE SUBSTITUTION WITH DEFINED CONFIGURATION ON THE THERMAL STABILTIY AND CONFORMATION OF A DNA DUPLEX

Substitution of one non-bridging oxygen in a natural phosphodiester internucleotide linkage with a borano (-BH3) group results in a chiral phosphorus center in boranophosphate. UV thermal melting profiles were recorded for DNA duplexes formed between a DNA 9-mer with either an Sp or a Rp boranophosphate linkage in the middle and the complementary DNA 9-mer, as well as for their unmodified parent duplex. The thermal stability of the DNA duplexes was in the order of normal > Sp borano > Rp borano. CD spectra of all three duplexes exhibited typical B-DNA profile, which closely resembled each other.     

The effect of a single boranophosphate substitution with defined configuration on the thermal stability and conformation of a DNA duplex

Substitution of one non-bridging oxygen in a natural phosphodiester internucleotide linkage with a borano (-BH3) group results in a chiral phosphorus center in boranophosphate. UV thermal melting profiles were recorded for DNA duplexes formed between a DNA 9-mer with either an Sp or a Rp boranophosphate linkage in the middle and the complementary DNA 9-mer, as well as for their unmodified parent duplex. The thermal stability of the DNA duplexes was in the order of normal > Sp borano > Rp borano. CD spectra of all three duplexes exhibited typical B-DNA profile, which closely resembled each other.     

The Synthesis and Properties of Oligodeoxyribonucleotides with Single Mono- and Diphosphoryldithio Internucleotide Links

The synthesis of oligodeoxyribonucleotides bearing mono- and diphosphoryldisulfide internucleotide links was optimized. Oligonucleotide 3"-phosphorothioates were modified using the thiophosphoryl–disulfide exchange with preactivated 5"-deoxy-5"-mercaptooligonucleotides or 5"-phosphorothioate derivatives both with and without a complementary template. The lack of template was shown to differently affect the product ratio (homo- and heterodimers) in the reactions of mono- and diphosphoryldisulfide-containing oligonucleotides. A replacement of one natural phosphodiester bond in 15–16-mer duplexes by a mono- or diphosphoryldisulfide group causes a slight thermal destabilization of the corresponding duplex. The disulfide recombination of the resulting compounds was studied.     

Lna (Locked Nucleic Acid)

Abstract

LNA (Locked Nucleic Acid) forms duplexes with complementary DNA, RNA or LNA with unprecedented thermal affinities. CD spectra show that duplexes involving fully modified LNA (especially LNA:RNA) structurally resemble an A-form RNA:RNA duplex. NMR examination of an LNA:DNA duplex confirm the 3′-endo conformation of an LNA monomer. Recognition of double-stranded DNA is demonstrated suggesting strand invasion by LNA. Lipofectin-mediated efficient delivery of LNA into living human breast cancer cells has been accomplished.     

Synthesis and hybridization of 2′-O-methyl-RNAs incorporating 2′-O-carbamoyluridine and unique participation of the carbamoyl group in U–G base pair

2′-O-Carbamoyluridine (U_cm) was synthesized and incorporated into DNAs and 2′-O-Me-RNAs. The oligonucleotides incorporating U_cm formed less stable duplexes with their complementary and U_cm–U, U_cm–C single-base mismatched DNAs and RNAs in comparison with those without the carbamoyl group. On the contrary, the T_m analyses revealed that the duplexes with a mismatched U_cm–G base pair showed almost the same thermostability as the corresponding unmodified duplexes. Molecular dynamics (MD) simulations of the U_cm-modified 2′-O-Me-RNA/RNA duplexes with U_cm–G mismatched base pair suggested that the carbamoyl group could participate in the U_cm–G base pair by an additional intermolecular hydrogen bond between the carbamoyl oxygen and the H2 of the guanine base.     

Oligonucleotide derivatives in nucleic acid hybridization analysis. III. Synthesis and investigation of properties of oligonucleotides, bearing bifunctional non-nucleotide insertion

Non-nucleotide phosporamidites were synthetized, having a branching backbone with different positions for functional groups. Phosphoramidite monomers obtained contain intercalator moiety, 6-chloro-2-methoxyacridine, and additional hydroxyl residue protected with dimethoxytrityl group or with the tert-butyldimethylsilyl group for post-synthetic modification. Oligothymidilates containing one or more modified units in different positions of the sequence were synthesized. The melting point and thermodynamic parameters of the formation of complementary duplexes formed by modified oligonucleotides were defined (change in enthalpy and entropy). The introduction of intercalating residue causes a significant stabilization of DNA duplexes. It is shown that the efficiency of the fluorescence of acridine residue in the oligonucleotide conjugate significantly changes upon hybridization with DNA.     

Oligonucleotide Conjugates Designed for Discriminative Hybridization at Physiological Temperature

Abstract

We report the synthesis of oligonucleotide conjugates engineered to allow discriminative hybridization at temperatures around physiological. Two types of structural modifications were introduced: 1) internal oligomethylene and oligoethylene glycol spacers, and 2) terminal phenazinium residues. The thermal denaturation behaviour of the complexes formed by these oligonucleotide conjugates with a target sequence is compared to that of natural duplexes. We observed a lowering of the T_m of the duplexes formed by the internal modified oligonucleotides, whilst the terminal phenazinium residues enhance their stability. The effect of the spacers is modulated by their length and hydrophobic or hydrophilic nature. Alkylating substituents, which modify the target DNA strand on hybridization, were introduced on all conjugates, and the target cleavage obtained after piperidine treatment used as a further indicator of hybridization.     

Watson-Crick base-pairing properties of bicyclo-DNA.

A series of sequences of the DNA analog bicyclo-DNA, 6-12 nucleotides in length and containing all four natural nucleobases, were prepared and their Watson-Crick pairing properties with complementary RNA and DNA, as well as in its own series, were analyzed by UV-melting curves and CD-spectroscopy. The results can be summarized as follows: bicyclo-DNA forms stable Watson-Crick duplexes with complementary RNA and DNA, the duplexes with RNA generally being more stable than those with DNA. Pyrimidine-rich bicyclo-DNA sequences form duplexes of equal or slightly increased stability with DNA or RNA, whereas purine-rich sequences show decreased affinity to complementary DNA and RNA when compared with wild-type (DNA-DNA, DNA-RNA) duplexes. In its own system, bicyclo-DNA prefers antiparallel strand alignment and strongly discriminates for base mismatches. Duplexes are always inferior in stability compared with the natural ones. A detailed analysis of the thermodynamic properties was performed with the sequence 5'-GGATGGGAG-3'x 5'-CTCCCATCC-3' in both backbone systems. Comparison of the pairing enthalpy and entropy terms shows an enthalpic advantage for DNA association (delta deltaH = -18 kcal x (mol)-1)) and an entropic advantage for bicyclo-DNA association (delta deltaS = 49 cal x K(-1) x mol(-1), leading to a delta deltaG 25 degrees C of -3.4 kcal x mol(-1) in favor of the natural duplex. The salt dependence of Tm for this sequence is more pronounced in the case of bicyclo-DNA due to increased counter ion screening from the solvent. Furthermore bicyclo-DNA sequences are more stable towards snake venom phosphodiesterase by a factor of 10-20, and show increased stability in fetal calf serum by a factor of 8 compared with DNA.     

Influence of 5-N-carboxamide modifications on the thermodynamic stability of oligonucleotides

We have recently shown that the incorporation of modified nucleotides such as 5-N-carboxamide-deoxyuridines into random nucleic acid libraries improves success rates in SELEX experiments and facilitates the identification of ligands with slow off-rates. Here we report the impact of these modifications on the thermodynamic stability of both duplexes and intramolecular ‘single-stranded’ structures. Within duplexes, large, hydrophobic naphthyl groups were destabilizing relative to the all natural DNA duplex, while the hydrophilic groups exhibited somewhat improved duplex stability. All of the significant changes in stability were driven by opposing contributions from the enthalpic and entropic terms. In contrast, both benzyl and naphthyl modifications stabilized intramolecular single-stranded structures relative to their natural DNA analogs, consistent with the notion that intramolecular folding allows formation of novel, stabilizing hydrophobic interactions. Imino proton NMR data provided evidence that elements of the folded structure form at temperatures well below the T_m, with a melting transition that is distinctly less cooperative when compared to duplex DNA. Although there are no data to suggest that the unmodified DNA sequences fold into structures similar to their modified analogs, this still represents clear evidence that these modifications impart thermodynamic stability to the folded structure not achievable with unmodified DNA.