New Strategies for the Chemical Synthesis of Biologically Important Nucleic Acid Derivatives

Abstract

This paper describes general methods for the synthesis of N-phosphorylated ribonucleosides and oligonucleotides containing a 2′-O-phosphorylated or 2′-O-thiophosphorylated ribonucleoside. The NMR-based conformational analysis and computational molecular dynamics simulation of the 2′-O-phosphorylated ribonucleoside residue in such modified oligonucleotides suggested that the ribose residue existed preferentially in a C2′-endo conformation. It was also found that simple heating of 2′-O-phosphorylated oligonucleotides resulted in rapid dethiophosphorylation.     

Serum Interferon-Related MicroRNAs as Biomarkers to Predict the Response to Interferon Therapy in Chronic Hepatitis C Genotype 4

MicroRNAs are messengers during interferon-virus interplay and are involved in antiviral immunity, however, little is known about interferon-related microRNAs regarding their detection in serum and their potential use as non-invasive diagnostic and prognostic biomarkers in chronic hepatitis C (CHC). To elucidate some of the molecular aspects underlying failure of pegylated interferon-α/ribavirin therapy, we investigated pretreatment expression profiles of seven selected interferon-related microRNAs (miR-146a, miR-34a, miR-130a, miR-19a, miR-192, miR-195, and miR-296) by quantitative RT-PCR custom array technology in serum of Egyptian CHC genotype 4 patients and whether their pretreatment levels would predict patient response to the combination therapy. One hundred and six CHC patients and forty matched healthy controls were included. Patients were divided into sustained virological response (SVR) and non-responder (NR) groups. Serum miR-34a, miR-130a, miR-19a, miR-192, miR-195, and miR-296 were upregulated, whereas serum miR-146a was downregulated in CHC compared to controls. Significant correlations were found between expression levels of studied microRNAs and also with clinical data. Pretreatment levels of miR-34a, miR-130a, and miR-195 were significantly higher, whereas miR-192 and miR-296 levels were significantly lower in SVR than NR patients. miR-19a and miR-146a levels were not significantly different between the two groups. miR-34a was superior to differentiate CHC from controls, whereas miR-296 was superior to discriminate SVR from NR patients by receiver operating characteristic analysis. Multivariate logistic analysis revealed miR-34a and miR-195 as independent predictors for SVR and miR-192 as an independent variable for non-response. In conclusion, pretreatment expression profiles of five interferon-related microRNAs are associated with treatment outcome in CHC. Of these, miR-34a, miR-195, and miR-192 could predict treatment response. The profiling results could be used as novel non-invasive diagnostic and prognostic pharmacogenetic biomarkers for treatment personalization in CHC and could help to identify new microRNA-based antivirals.     

A method of gentle hydration to prepare oil-free giant unilamellar vesicles that can confine enzymatic reactions

We report a new and improved method to prepare, by gentle hydration of lipid films, oil-free giant unilamellar vesicles (GUVs), in which enzymatic reactions can be encapsulated. The traditional method of gentle hydration requires very low concentrations of metal ions, whereas enzymatic reactions generally require mono- and divalent metal ions at physiological concentrations. In order to improve the production of oil-free GUVs that can confine enzymatic reactions, we developed a novel method also based on gentle hydration, but in which the precursor lipid film was doped with both 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (PEGylated lipid) and sugar. Close examination of the size, shape, and lamellarity of vesicles prepared in this manner demonstrated that the process improves the production of oil-free GUVs even at low temperatures and physiological salt concentrations. PEGylated lipid and sugar were found to synergistically improve GUV formation. Finally, we demonstrate the successful enzymatic synthesis of RNA within oil-free GUVs that were prepared on ice.     

Heat-resistant DNA tile arrays constructed by template-directed photoligation through 5-carboxyvinyl-2′-deoxyuridine

Template-directed DNA photoligation has been applied to a method to construct heat-resistant two-dimensional (2D) DNA arrays that can work as scaffolds in bottom-up assembly of functional biomolecules and nano-electronic components. DNA double-crossover AB-staggered (DXAB) tiles were covalently connected by enzyme-free template-directed photoligation, which enables a specific ligation reaction in an extremely tight space and under buffer conditions where no enzymes work efficiently. DNA nanostructures created by self-assembly of the DXAB tiles before and after photoligation have been visualized by high-resolution, tapping mode atomic force microscopy in buffer. The improvement of the heat tolerance of 2D DNA arrays was confirmed by heating and visualizing the DNA nanostructures. The heat-resistant DNA arrays may expand the potential of DNA as functional materials in biotechnology and nanotechnology.     

Synthesis and properties of 2'-O-methyl-2-thiouridine and oligoribonucleotides containing 2'-O-methyl-2-thiouridine

A new method for the synthesis of 2'-O-methyl-2-thiouridine (s2Um) found in thermophilic bacterial tRNA was developed. Structural properties of s2Um and s2Um(p)U were studied by using 1H NMR spectroscopy. A modified nonaribonucleotide (RNA*: 5'-CGUUs2UmUUGC-3') was synthesized to study the base-recognition ability of s2Um in formation of RNA-RNA and RNA DNA duplexes. The UV melting experiments revealed that RNA*-RNA and RNA*-DNA duplexes having an s2U-A base pair are more stable than those having a U-A base pair. On the contrary, the thermal stability of RNA*-RNA and RNA*-DNA duplexes having an s2U-G wobble base pair was much lower than that of the unmodified duplexes having a natural U-G base pair. It is concluded that s2Um has higher selectivity toward A over G than unmodified U.     

Serum MicroRNAs as Potential Biomarkers for Early Diagnosis of Hepatitis C Virus-Related Hepatocellular Carcinoma in Egyptian Patients

Circulating microRNAs are deregulated in liver fibrosis and hepatocellular carcinoma (HCC) and are candidate biomarkers. This study investigated the potential of serum microRNAs; miR-19a, miR-296, miR-130a, miR-195, miR-192, miR-34a, and miR-146a as early diagnostic biomarkers for hepatitis C virus (HCV)-related HCC. As how these microRNAs change during liver fibrosis progression is not clear, we explored their serum levels during fibrosis progression in HCV-associated chronic liver disease (CLD) and if they could serve as non-invasive biomarkers for fibrosis progression to HCC. 112 Egyptian HCV-HCC patients, 125 non-malignant HCV-CLD patients, and 42 healthy controls were included. CLD patients were subdivided according to Metavir fibrosis-scoring. Serum microRNAs were measured by qRT-PCR custom array. Serum microRNAs were deregulated in HCC versus controls, and except miR-130a, they were differentially expressed between HCC and CLD or late fibrosis (F3-F4) subgroup. Serum microRNAs were not significantly different between individual fibrosis-stages or between F1-F2 (early/moderate fibrosis) and F3-F4. Only miR-19a was significantly downregulated from liver fibrosis (F1-F3) to cirrhosis (F4) to HCC. Individual microRNAs discriminated HCC from controls, and except miR-130a, they distinguished HCC from CLD or F3-F4 patients by receiver-operating-characteristic analysis. Multivariate logistic analysis revealed a panel of four microRNAs (miR-19a, miR-195, miR-192, and miR-146a) with high diagnostic accuracy for HCC (AUC = 0.946). The microRNA panel also discriminated HCC from controls (AUC = 0.949), CLD (AUC = 0.945), and F3-F4 (AUC = 0.955). Studied microRNAs were positively correlated in HCC group. miR-19a and miR-34a were correlated with portal vein thrombosis and HCC staging scores, respectively. In conclusion, studied microRNAs, but not miR-130a, could serve as potential early biomarkers for HCC in high-risk groups, with miR-19a as a biomarker for liver fibrosis progression to cirrhosis to HCC. We identified a panel of four serum microRNAs with high accuracy in HCC diagnosis. Additional studies are required to confirm this panel and test its prognostic significance.     

Synthesis of Artificially Bent Oligonucleotides by Incorporation of Conformationally Rigid 5′-Cyclouridylic Acid Derivatives

Abstract

This paper describes the design and synthesis of a conformationally rigid dimer building block Umpc3Um having a propylene bridge linked between the uracil 5-position and 5′-phosphate group of pUm. Oligonucleotides incorporating the dimer unit with either the Sp or Rp configuration were synthesized by use of the phosphoramidite approach. The conformational properties of the dimer units and these oligonucleotides were studied in detail.     

Computational evaluation of intermolecular interactions of a universal base 3-nitropyrrole in stacked dimers and DNA duplexes

The stacking interactions between a universal base of 3-nitropyrrole (3NP) and four canonical nucleobases were studied by means of ab initio molecular orbital calculations. The stabilities of the complexes are comparable to those of the stacked dimers of canonical bases reported previously. The detailed analysis of the interaction energies revealed the importance of the dipole-dipole interaction included in the Hartree-Fock terms to determine the geometry dependence of the stacking energies. It was also clarified that the dispersion energies included in the electron-correlation terms were essential to obtain adequate stabilities. The contribution of the nitro group was evaluated by the comparative studies of pyrrole and 3NP. The increased molecular dipole moment and surface are expected to account for the enhancement of the stability of the stacked dimers containing 3NP. The force field parameters required for calculation of the molecular mechanics of 3NP were obtained for 3NP on the basis of these molecular orbital calculations. The energy-minimized structures obtained by the molecular mechanics calculations of 3NP accorded with those obtained by the molecular orbital calculations described above. A DNA duplex structure containing 3NP-A, 3NP-T, or 3NP-C was calculated by use of these force field parameters. In the case of 3NP-A, the computationally calculated structure was in good agreement with that previously determined by use of (1)H-NMR except for the orientation of the nitro group.     

Computational Evaluation of Intermolecular Interactions of a Universal Base 3-Nitropyrrole in Stacked Dimers and DNA Duplexes

Abstract

The stacking interactions between a universal base of 3-nitropyrrole (3NP) and four canonical nucleobases were studied by means of ab initio molecular orbital calculations. The stabilities of the complexes are comparable to those of the stacked dimers of canonical bases reported previously. The detailed analysis of the interaction energies revealed the importance of the dipole-dipole interaction included in the Hartree-Fock terms to determine the geometry dependence of the stacking energies. It was also clarified that the dispersion energies included in the electron-correlation terms were essential to obtain adequate stabilities. The contribution of the nitro group was evaluated by the comparative studies of pyrrole and 3NP. The increased molecular dipole moment and surface are expected to account for the enhancement of the stability of the stacked dimers containing 3NP. The force field parameters required for calculation of the molecular mechanics of 3NP were obtained for 3NP on the basis of these molecular orbital calculations. The energy-minimized structures obtained by the molecular mechanics calculations of 3NP accorded with those obtained by the molecular orbital calculations described above. A DNA duplex structure containing 3NP-A, 3NP-T, or 3NP-C was calculated by use of these force field parameters. In the case of 3NP-A, the computationally calculated structure was in good agreement with that previously determined by use of ¹H-NMR except for the orientation of the nitro group.