Oligonucleotide analogs with peptide internucleotide linkages

Oligonucleotide analogs containing one or a few glycine, L-, and D-alanine or L-and D-phenylalanine residues instead of phosphodiesterinternucleotide linkages were synthesized. The stability of the duplexes formed by modified oligonucleotides and their wildtype complements was studied. Oligonucleotides with D-alanine residues in internucleotide linkages form duplexes more stable than native ones (ΔT(m) +0.2 °C per modification), whereas other modifications destabilize the duplexes.     

Investigating oligonucleotide hybridization at subnanomolar level by surface plasmon resonance biosensor method

We have optimized surface plasmon resonance (SPR) biosensor technology for a rapid, direct, and low-consumption label-free multianalyte screening of synthetic oligonucleotides (ONs) with modified internucleotide linkages potentially applicable in antisense therapy. Monitoring of the ONs hybridization is based on the formation of complex between the natural oligonucleotide probe immobilized on the sensor surface and the ON in solution in contact with the sensor surface. An immobilization chemistry utilizing the streptavidin-biotin interaction was employed to obtain desired ligand density and high hybridization efficiency. It was demonstrated that the sensor is capable of detecting complementary 23-mer ONs in concentrations as low as 0.1 nM with high specificity and reproducibility.     

Site specific functionalization of oligonucleotides for attaching two different reporter groups.

The synthesis of an oligonucleotide functionalized to attach two different reporter groups at specific internucleotide linkages is described. To incorporate the amine specific reporter group the internucleotide linkage is modified to phosphoramidate (N-1-aminoalkyl) and for a thiol specific reporter group the internucleotide linkage is modified to a phosphorothioate diester. The synthetic cycle for introducing the modified internucleotide linkages at specific sites can be carried out using an automated DNA synthesizer. Combination of reporter groups have been attached successfully.     

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.     

Specificities of rabbit anti-(2',5')oligoadenylate antibodies towards phosphorothioate and seco analogs of oligoadenylates.

Rabbit antibodies to 2',5'-linked triadenylate were prepared by immunization with (2',5')A3 conjugated via the 2'3'-levulinic group, (2'5')A3-Lev, to BSA. New radioimmunoassay for (2',5')oligoadenylates was developed using 125I thyrosine labeled derivative of (2',5')A3-Lev. Reactivity of antibodies with phosphorothioate and seco analogs of oligoadenylates was studied. It was found that (i) stereospecific substitution of the diastereotopic oxygens with sulfur in the internucleotide phosphodiester linkages changes the immunoreactivity of such analogs; (ii) the seco analogs of oligoadenylates display in some cases a rather high reactivity.     

The Histone H4 Tail Regulates the Conformation of the ATP-Binding Pocket in the SNF2h Chromatin Remodeling Enzyme

The chromatin remodeling complex ACF helps establish the appropriate nucleosome spacing for generating repressed chromatin states. ACF activity is stimulated by two defining features of the nucleosomal substrate: a basic patch on the histone H4 N-terminal tail and the specific length of flanking DNA. However, the mechanisms by which these two substrate cues function in the ACF remodeling reaction is not well understood. Using electron paramagnetic resonance spectroscopy with spin-labeled ATP analogs to probe the structure of the ATP active site under physiological solution conditions, we identify a closed state of the ATP-binding pocket that correlates with ATPase activity. We find that the H4 tail promotes pocket closure. We further show that ATPase stimulation by the H4 tail does not require a specific structure connecting the H4 tail and the globular domain. In the case of many DNA helicases, closure of the ATP-binding pocket is regulated by specific DNA substrates. Pocket closure by the H4 tail may analogously provide a mechanism to directly couple substrate recognition to activity. Surprisingly, the flanking DNA, which also stimulates ATP hydrolysis, does not promote pocket closure, suggesting that the H4 tail and flanking DNA may be recognized in different reaction steps.     

Stereoconfiguration markedly affects the biochemical and biological properties of phosphorothioate analogs of 2-5A core, (A2'p5')2A

The diester bonds of phosphorothioate trimer analogs of (A2'p5')2A (2-5A core) of the Sp stereoconfiguration were found to be extremely stable to hydrolysis by both serum and cellular phosphodiesterases. The corresponding Rp isomers, although still more stable than parent ppp(A2'p5')2A (2-5A), were significantly more susceptible to enzymatic hydrolysis than were the Sp isomers. Utilization of these novel 2-5A trimer isomers containing various combinations of Sp or Rp configurations at the internucleotidic phosphorothioate linkages revealed a further specificity of this enzymatic hydrolysis. Thus, the stereoconfiguration of the bond adjacent to the one undergoing hydrolysis influenced the rate of enzymatic hydrolysis, as well as did the chain length of the oligomer. The most stable trimer analog, which contained both internucleotide phosphorothioate linkages of the Sp configuration, had a half-life of 30 days in serum, which is a 1500-fold increase over that of parent 2-5A core. This is the first report on biochemical stability of an oligonucleotide containing more than one phosphorothioate linkage of the Sp configuration and is the first demonstration that a phosphorothioate internucleotide bond of the Sp configuration can increase the enzymatic stability of an adjacent phosphorothioate bond. In marked contrast to all previous 2-5A core analogs of increased stability, the activity (antiproliferative and antiviral) of the stable phosphorothioate 2-5A core analogs was obtained with the intact trimer, i.e., it was not attributed to antimetabolite degradation products.     

Differences in the Detection of BrdU/EdU Incorporation Assays Alter the Calculation for G1, S,and G2 Phases of the Cell Cycle in Trypanosomatids

Trypanosomatids are the etiologic agents of various infectious diseases in humans. They diverged early during eukaryotic evolution and have attracted attention as peculiar models for evolutionary and comparative studies. Here, we show a meticulous study comparing the incorporation and detection of the thymidine analogs BrdU and EdU in Leishmania amazonensis, Trypanosoma brucei, and Trypanosoma cruzi to monitor their DNA replication. We used BrdU‐ and EdU‐incorporated parasites with the respective standard detection approaches: indirect immunofluorescence to detect BrdU after standard denaturation (2 M HCl) and “click” chemistry to detect EdU. We found a discrepancy between these two thymidine analogs due to the poor detection of BrdU, which is reflected on the estimative of the duration of the cell cycle phases G1, S, and G2. To solve this discrepancy, we increase the exposure of incorporated BrdU using different concentrations of HCl. Using a new value for HCl concentration, we re‐estimated the phases G1, S, G2 + M, and cytokinesis durations, confirming the values found by this approach using EdU. In conclusion, we suggest that the studies using BrdU with standard detection approach, not only in trypanosomatids but also in others cell types, should be reviewed to ensure an accurate estimation of DNA replication monitoring.     

Synthesis and selective cleavage of oligodeoxyribonucleotides containing non-chiral internucleotide phosphoramidate linkages.

Oligodeoxynucleotides containing phosphoramidate internucleotide links 3'-OP(O)NH-5' have been prepared using standard solid phase phosphoramidite techniques. For the incorporation of the phosphoramidate linkages we have used monomer as well as dimer building blocks. With the monomer 3'-phosphoramidite building blocks, which are derived from 5'-amino-2',5'-dideoxynucleosides, it is possible to incorporate phosphoramidate links into specific positions within an oligodeoxynucleotide. Furthermore the synthesis of several dinucleoside phosphate derivatives which are linked by phosphoramidate bonds are described. The internucleotide phosphoramidate linkage was performed using the Staudinger reaction followed by a Michaelis-Arbuzov type transformation. After 3'-phosphitylation these dinucleosides are compatible with the current phosphoramidite methodology of oligodeoxynucleotide synthesis.     

Hoogsteen-paired homopurine [RP-PS]-DNA and homopyrimidine RNA strands form a thermally stable parallel duplex

Homopurine deoxyribonucleoside phosphorothioates possessing all internucleotide linkages of R(P) configuration form a duplex with an RNA or 2'-OMe-RNA strand with Hoogsteen complementarity. The duplexes formed with RNA templates are thermally stable at pH 5.3, while those formed with a 2'-OMe-RNA are stable at neutrality. Melting temperature and fluorescence quenching experiments indicate that the strands are parallel. Remarkably, these duplexes are thermally more stable than parallel Hoogsteen duplexes and antiparallel Watson-Crick duplexes formed by unmodified homopurine DNA molecules of the same sequence with corresponding RNA templates.     

Synthesis and properties of triazole-linked RNA

RNA oligonucleotides having triazole linkages between uridine and adenosine nucleosides have been prepared and studied using spectroscopic techniques. UV melting and CD showed that triazole strongly destabilized RNA duplex (7-14 °C per modification). NMR data suggested that, despite relative flexibility around the modified linkage, all base pairs were formed.     

Progress toward the synthesis of nonionic oligonucleotide analogues with sulfonate and sulfonamide internucleotide linkages.

The synthesis of building blocks for the preparation of nonionic oligonucleotide analogues with sulfonate and sulfonamide internucleotide linkages is described. Coupling conditions for the conversion of several of these monomers to dimers are also described.     

Interdomain Ca effects in Escherichia coli α-haemolysin: Ca binding to the C-terminal domain stabilizes both C- and N-terminal domains

α-Haemolysin (HlyA) is a toxin secreted by pathogenic Escherichia coli, whose lytic activity requires submillimolar Ca²⁺ concentrations. Previous studies have shown that Ca²⁺ binds within the Asp and Gly rich C-terminal nonapeptide repeat domain (NRD) in HlyA. The presence of the NRD puts HlyA in the RTX (Repeats in Toxin) family of proteins. We tested the stability of the whole protein, the amphipathic helix domain and the NRD, in both the presence and absence of Ca²⁺ using native HlyA, a truncated form of HlyAΔN601 representing the C-terminal domain, and a novel mutant HlyA W914A whose intrinsic fluorescence indicates changes in the N-terminal domain. Fluorescence and infrared spectroscopy, tryptic digestion, and urea denaturation techniques concur in showing that calcium binding to the repeat domain of α-haemolysin stabilizes and compacts both the NRD and the N-terminal domains of HlyA. The stabilization of the N-terminus through Ca²⁺ binding to the C-terminus reveals long-range inter-domain structural effects. Considering that RTX proteins consist, in general, of a Ca²⁺-binding NRD and separate function-specific domains, the long-range stabilizing effects of Ca²⁺ in HlyA may well be common to other members of this family.     

Novel antisense oligonucleotides containing hydroxamate linkages: targeted iron-triggered chemical nucleases

Antisense oligonucleotides with iron binding hydroxamate linkages are designed to act as sequence-selective cleaving agents of complementary nucleic acids through Fenton chemistry. Oligothymidylate analogs with hydroxamate linkages were efficiently synthesized from coupling of nucleoside intermediates, activated as p-nitrophenyl carbonates, with hydroxylamine derivatized nucleosides. Iron binding studies showed that hydroxamate linked oligonucleotides are effective iron chelators when there are three nonadjacent internucleosidic hydroxamate linkages available in the same oligonucleotide molecule. However, analysis of the CD spectra of an oligothymidylate 16mer, which contained complete substitution of all phosphates with hydroxamates, indicated that the hydroxamate linkage was too rigid to allow the analog to base pair with the complementary DNA d(A₁₆). Syntheses of mix-linked thymidine oligomers with up to three hydroxamate linkages incorporated in the center of the sequence are also reported. Iron binding of the thymidine oligomer with hydroxamate linkages was confirmed by matrix assisted laser desorption mass spectrometry analysis. Nuclease stability assays showed that the modified oligonucleotides have enhanced resistance toward nuclease S1 (endonuclease) compared to natural oligonucleotides. A thymidine 16mer with three hydroxamate linkages incorporated in the center of the sequence was shown to be able to bind with both iron and its complementary polyA strand. A small destablizing effect was observed when the phosphodiester linkage was changed to the hydroxamate linkage. Under Fenton chemistry conditions, this novel iron binding oligothymidylate analog cleaved the complementary DNA strand sequence-selectively.     

Synthesis and Hybridization Properties of Oligonucleotides Containing (2 S ,3 R )-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.     

Nile Tilapia Neu3 sialidases: Molecular cloning,functional characterization and expression in Oreochromis niloticus

Mammalian Neu3 is a ganglioside specific sialidase. Gangliosides are involved in various physiological events such as cell growth, differentiation and diseases. Significance of Neu3 and gangliosides is still unclear in aquaculture fish species. To gain more insights of fish Neu3 sialidases, molecular cloning and characterization were carried out in tilapia (Oreochromis niloticus). A tilapia genome-wide search for orthologues of human NEU1, NEU2, NEU3 and NEU4 yielded eight putative tilapia sialidases, five of which were neu3-like and designated as neu3a, neu3b, neu3c, neu3d and neu3e. Among five neu3 genes, neu3a, neu3d and neu3e were amplified by PCR from adult fish brain cDNA with consensus sequences of 1227 bp, 1194 bp and 1155 bp, respectively. Multiple alignments showed conserved three Asp-boxes (SXDXGXTW), YRIP and VGPG motifs. The molecular weights for Neu3a, Neu3d and Neu3e were confirmed using immunoblotting analysis as 45.9 kDa, 44.4 kDa and 43.6 kDa, respectively. Lysate from neu3 genes transfected HEK293 cells showed sialidase activity in Neu3a towards ganglioside mix optimally at pH 4.6. Using pure gangliosides as substrates, highest sialidase activity for Neu3a was observed towards GD3 followed by GD1a and GM3, but not GM1. On the other hand, sialidase activities were not observed in Neu3d and Neu3e towards various sialoglycoconjugates. Indirect immunofluorescence showed that tilapia Neu3a and Neu3d are localized at the plasma membrane, while most Neu3e showed a cytosolic localization. RT-PCR analyses for neu3a showed significant expression in the brain, liver, and spleen tissues, while neu3d and neu3e showed different expression patterns. Based on these results, tilapia Neu3 exploration is an important step towards full understanding of a more comprehensive picture of Neu3 sub-family of proteins in fish.     

Evaluation of "click" binaphthyl chiral stationary phases by liquid chromatography

Two "click" binaphthyl chiral stationary phases were synthesized and evaluated by liquid chromatography. Their structures incorporate S-(-)-1,1'-binaphthyl moiety as the chiral selector and 1,2,3-triazole ring as the spacer. These chiral stationary phases (CSPs) allowed the efficient resolution for a wide range of racemic BINOL derivatives, particularly for nonpolar diether derivatives and 3-phenyl indolin-2-one analogs. The chromatographic data showed that the π-π interaction was crucial for enantiorecognition of these CSPs. Loss of enantioselectivity observed on CSP3, which are lacking the triazole ring linkage, indicated that the triazole ring linkage took part in the enantioseparation process, although it was remote from the chiral selector of the CSP. The substitution of the phenyl group at 6 and 6' positions can significantly improve the separation ability of the CSP. The chiral recognition mechanism was also investigated by tracking the elution orders and studying the thermodynamic parameters.     

Relationship between internucleotide linkage geometry and the stability of RNA

The inherent chemical instability of RNA under physiological conditions is primarily due to the spontaneous cleavage of phosphodiester linkages via intramolecular transesterification reactions. Although the protonation state of the nucleophilic 2'-hydroxyl group is a critical determinant of the rate of RNA cleavage, the precise geometry of the chemical groups that comprise each internucleotide linkage also has a significant impact on cleavage activity. Specifically, transesterification is expected to be proportional to the relative in-line character of the linkage. We have examined the rates of spontaneous cleavage of various RNAs for which the secondary and tertiary structures have previously been modeled using either NMR or X-ray crystallographic data. Rate constants determined for the spontaneous cleavage of different RNA linkages vary by almost 10,000-fold, most likely reflecting the contribution that secondary and tertiary structures make towards the overall chemical stability of RNA. Moreover, a correlation is observed between RNA cleavage rate and the relative in-line fitness of each internucleotide linkage. One linkage located within an ATP-binding RNA aptamer is predicted to adopt most closely the ideal conformation for in-line attack. This linkage has a rate constant for transesterification that is approximately 12-fold greater than is observed for an unconstrained linkage and was found to be the most labile among a total of 136 different sites examined. The implications of this relationship for the chemical stability of RNA and for the mechanisms of nucleases and ribozymes are discussed.     

Cationic phosphoramidate alpha-oligonucleotides efficiently target single-stranded DNA and RNA and inhibit hepatitis C virus IRES-mediated translation

A potential means to improve the efficacy of steric-blocking antisense oligonucleotides (ON) is to increase their affinity for a target RNA. The grafting of cationic amino groups to the backbone of the ON is one way to achieve this, as it reduces the electrostatic repulsion between the ON and its target. We have examined the duplex stabilising effects of introducing cationic phosphoramidate internucleoside linkages into ON with a non-natural α-anomeric configuration. Cationic α-ON bound with high affinity to single-stranded DNA and RNA targets. Duplex stabilisation was proportional to the number of cationic modifications, with fully cationic ON having particularly high thermal stability. The average stabilisation was greatly increased at low ionic strength. The duplex formed between cationic α-ON and their RNA targets were not substrates for RNase H. The penalty in T_m inflicted by a single mismatch, however, was high; suggesting that they are well suited as sequence-specific, steric-blocking, antisense agents. Using a well-described target sequence in the internal ribosome entry site of the human hepatitis C virus, we have confirmed this potential in a cell-free translation assay as well as in a whole cell assay. Interestingly, no vectorisation was necessary for the cationic α-ON in cell culture.     

Synthesis and evaluation of a novel quinoline-triazole analogs for antitubercular properties via molecular hybridization approach

Towards a quest for establishing new antitubercular agents, we have designed new quinoline–triazole hybrid analogs in a six-step reaction sequence involving versatile reactions like Vilsmeier–Haack and click reaction protocol. The design is based on the structural modification of bedaquiline moiety and involves molecular hybridization approach. The structure of the synthesized product was elucidated by single crystal X-ray diffraction study. The synthesized target compounds were screened for their antitubercular activity against Mycobacterium bovis. Interestingly, two compounds of the series (8d and 8m) showed significant inhibition with MIC of 31.5 and 34.8 μM. Compounds bearing 3-fluoro phenyl and n-octyl groups on the 1,2,3-triazole ring emerged as the most potent leads among the compounds tested. Further these hit compounds were also screened for their cytotoxic effect on human embryonic kindey 293 (HEK293) cells and other cancer cell lines such as HeLa (Cervical), PC3 (Prostate), Panc-1 (Pancreatic) and SKOV3 (Ovarian) indicating to be safer with the minimal cytotoxicity.