Synthesis of 1,4-azaphosphinine nucleosides and evaluation as inhibitors of human cytidine deaminase and APOBEC3A.

Nucleoside and polynucleotide cytidine deaminases (CDAs), such as CDA and APOBEC3, share a similar mechanism of cytosine to uracil conversion. In 1984, phosphapyrimidine riboside was characterised as the most potent inhibitor of human CDA, but the quick degradation in water limited the applicability as a potential therapeutic. To improve stability in water, we synthesised derivatives of phosphapyrimidine nucleoside having a CH2 group instead of the N3 atom in the nucleobase. A charge-neutral phosphinamide and a negatively charged phosphinic acid derivative had excellent stability in water at pH 7.4, but only the charge-neutral compound inhibited human CDA, similar to previously described 2'-deoxyzebularine (Ki = 8.0 ± 1.9 and 10.7 ± 0.5 µM, respectively). However, under basic conditions, the charge-neutral phosphinamide was unstable, which prevented the incorporation into DNA using conventional DNA chemistry. In contrast, the negatively charged phosphinic acid derivative was incorporated into DNA instead of the target 2'-deoxycytidine using an automated DNA synthesiser, but no inhibition of APOBEC3A was observed for modified DNAs. Although this shows that the negative charge is poorly accommodated in the active site of CDA and APOBEC3, the synthetic route reported here provides opportunities for the synthesis of other derivatives of phosphapyrimidine riboside for potential development of more potent CDA and APOBEC3 inhibitors.

Seven-membered ring nucleobases as inhibitors of human cytidine deaminase and APOBEC3A.

The APOBEC3 (APOBEC3A-H) enzyme family as a part of the human innate immune system deaminates cytosine to uracil in single-stranded DNA (ssDNA) and thereby prevents the spread of pathogenic genetic information. However, APOBEC3-induced mutagenesis promotes viral and cancer evolution, thus enabling the progression of diseases and development of drug resistance. Therefore, APOBEC3 inhibition offers a possibility to complement existing antiviral and anticancer therapies and prevent the emergence of drug resistance, thus making such therapies effective for longer periods of time. Here, we synthesised nucleosides containing seven-membered nucleobases based on azepinone and compared their inhibitory potential against human cytidine deaminase (hCDA) and APOBEC3A with previously described 2'-deoxyzebularine (dZ) and 5-fluoro-2'-deoxyzebularine (FdZ). The nanomolar inhibitor of wild-type APOBEC3A was obtained by the incorporation of 1,3,4,7-tetrahydro-2H-1,3-diazepin-2-one in the TTC loop of a DNA hairpin instead of the target 2'-deoxycytidine providing a Ki of 290 ± 40 nM, which is only slightly weaker than the Ki of the FdZ-containing inhibitor (117 ± 15 nM). A less potent but notably different inhibition of human cytidine deaminase (CDA) and engineered C-terminal domain of APOBEC3B was observed for 2'-deoxyribosides of the S and R isomers of hexahydro-5-hydroxy-azepin-2-one: the S-isomer was more active than the R-isomer. The S-isomer shows resemblance in the position of the OH-group observed recently for the hydrated dZ and FdZ in the crystal structures with APOBEC3G and APOBEC3A, respectively. This shows that 7-membered ring analogues of pyrimidine nucleosides can serve as a platform for further development of modified ssDNAs as powerful A3 inhibitors.

Design, Synthesis, and Evaluation of a Cross-Linked Oligonucleotide as the First Nanomolar Inhibitor of APOBEC3A.

Drug resistance is a major problem associated with anticancer chemo- and immunotherapies. Recent advances in the understanding of resistance mechanisms have revealed that enzymes of the APOBEC3 (A3) family contribute to the development of drug resistance in multiple cancers. A3 enzymes are polynucleotide cytidine deaminases that convert cytosine to uracil (C→U) in single-stranded DNA (ssDNA) and in this way protect humans against viruses and mobile retroelements. On the other hand, cancer cells use A3s, especially A3A and A3B, to mutate human DNA, and thus by increasing rates of evolution, cancer cells escape adaptive immune responses and resist drugs. However, as A3A and A3B are non-essential for primary metabolism, their inhibition opens up a strategy to augment existing anticancer therapies and suppress cancer evolution. To test our hypothesis that pre-shaped ssDNA mimicking the U-shape observed in ssDNA-A3 complexes can provide a better binder to A3 enzymes, a Cu(I)-catalyzed azide-alkyne cycloaddition was used to cross-link two distant modified nucleobases in ssDNA. The resultant cytosine-containing substrate, where the cytosine sits at the apex of the loop, was deaminated faster by the engineered C-terminal domain of A3B than a standard, linear substrate. The cross-linked ssDNA was converted into an A3 inhibitor by replacing the 2'-deoxycytidine in the preferred TCA substrate motif by 2'-deoxyzebularine, a known inhibitor of single nucleoside cytidine deaminases. This strategy yielded the first nanomolar inhibitor of engineered A3BCTD and wild-type A3A (Ki = 690 ± 140 and 360 ± 120 nM, respectively), providing a platform for further development of powerful A3 inhibitors.

Differential Inhibition of APOBEC3 DNA-Mutator Isozymes by Fluoro- and Non-Fluoro-Substituted 2'-Deoxyzebularine Embedded in Single-Stranded DNA.

The APOBEC3 (APOBEC3A-H) enzyme family is part of the human innate immune system that restricts pathogens by scrambling pathogenic single-stranded (ss) DNA by deamination of cytosines to produce uracil residues. However, APOBEC3-mediated mutagenesis of viral and cancer DNA promotes its evolution, thus enabling disease progression and the development of drug resistance. Therefore, APOBEC3 inhibition offers a new strategy to complement existing antiviral and anticancer therapies by making such therapies effective for longer periods of time, thereby preventing the emergence of drug resistance. Here, we have synthesised 2'-deoxynucleoside forms of several known inhibitors of cytidine deaminase (CDA), incorporated them into oligodeoxynucleotides (oligos) in place of 2'-deoxycytidine in the preferred substrates of APOBEC3A, APOBEC3B, and APOBEC3G, and evaluated their inhibitory potential against these enzymes. An oligo containing a 5-fluoro-2'-deoxyzebularine (5FdZ) motif exhibited an inhibition constant against APOBEC3B 3.5 times better than that of the comparable 2'-deoxyzebularine-containing (dZ-containing) oligo. A similar inhibition trend was observed for wild-type APOBEC3A. In contrast, use of the 5FdZ motif in an oligo designed for APOBEC3G inhibition resulted in an inhibitor that was less potent than the dZ-containing oligo both in the case of APOBEC3GCTD and in that of full-length wild-type APOBEC3G.

Inhibiting APOBEC3 Activity with Single-Stranded DNA Containing 2'-Deoxyzebularine Analogues.

APOBEC3 enzymes form part of the innate immune system by deaminating cytosine to uracil in single-stranded DNA (ssDNA) and thereby preventing the spread of pathogenic genetic information. However, APOBEC mutagenesis is also exploited by viruses and cancer cells to increase rates of evolution, escape adaptive immune responses, and resist drugs. This raises the possibility of APOBEC3 inhibition as a strategy for augmenting existing antiviral and anticancer therapies. Here we show that, upon incorporation into short ssDNAs, the cytidine nucleoside analogue 2'-deoxyzebularine (dZ) becomes capable of inhibiting the catalytic activity of selected APOBEC variants derived from APOBEC3A, APOBEC3B, and APOBEC3G, supporting a mechanism in which ssDNA delivers dZ to the active site. Multiple experimental approaches, including isothermal titration calorimetry, fluorescence polarization, protein thermal shift, and nuclear magnetic resonance spectroscopy assays, demonstrate nanomolar dissociation constants and low micromolar inhibition constants. These dZ-containing ssDNAs constitute the first substrate-like APOBEC3 inhibitors and, together, comprise a platform for developing nucleic acid-based inhibitors with cellular activity.

Selective inhibition of APOBEC3 enzymes by single-stranded DNAs containing 2'-deoxyzebularine.

To restrict pathogens, in a normal human cell, APOBEC3 enzymes mutate cytosine to uracil in foreign single-stranded DNAs. However, in cancer cells, APOBEC3B (one of seven APOBEC3 enzymes) has been identified as the primary source of genetic mutations. As such, APOBEC3B promotes evolution and progression of cancers and leads to development of drug resistance in multiple cancers. As APOBEC3B is a non-essential protein, its inhibition can be used to suppress emergence of drug resistance in existing anti-cancer therapies. Because of the vital role of APOBEC3 enzymes in innate immunity, selective inhibitors targeting only APOBEC3B are required. Here, we use the discriminative properties of wild-type APOBEC3A, APOBEC3B and APOBEC3G to deaminate different cytosines in the CCC-recognition motif in order to best place the cytidine analogue 2'-deoxyzebularine (dZ) in the CCC-motif. Using several APOBEC3 variants that mimic deamination patterns of wild-type enzymes, we demonstrate that selective inhibition of APOBEC3B in preference to other APOBEC3 constructs is feasible for the dZCC motif. This work is an important step towards development of in vivo tools to inhibit APOBEC3 enzymes in living cells by using short, chemically modified oligonucleotides.

Automated Solid-Phase Click Synthesis of Oligonucleotide Conjugates: From Small Molecules to Diverse N-Acetylgalactosamine Clusters.

We developed a novel technique for the efficient conjugation of oligonucleotides with various alkyl azides such as fluorescent dyes, biotin, cholesterol, N-acetylgalactosamine (GalNAc), etc. using copper-catalysed alkyne-azide cycloaddition on the solid phase and CuI·P(OEt)3 as a catalyst. Conjugation is carried out in an oligonucleotide synthesizer in fully automated mode and is coupled to oligonucleotide synthesis and on-column deprotection. We also suggest a set of reagents for the construction of diverse conjugates. The sequential double-click procedure using a pentaerythritol-derived tetraazide followed by the addition of a GalNAc or Tris-GalNAc alkyne gives oligonucleotide-GalNAc dendrimer conjugates in good yields with minimal excess of sophisticated alkyne reagents. The approach is suitable for high-throughput synthesis of oligonucleotide conjugates ranging from fluorescent DNA probes to various multi-GalNAc derivatives of 2'-modified siRNA.

Determination of Concentration of Amphiphilic Polymer Molecules on the Surface of Encapsulated Semiconductor Nanocrystals.

We present a method for the determination of the average number of polymer molecules on the surface of A(II)B(VI) luminescent core-shell nanocrystals (CdSe/ZnS, ZnSe/ZnS quantum dots, and CdS/ZnS nanorods) encapsulated with amphiphilic polymer. Poly(maleic anhydride-alt-1-tetradecene) (PMAT) was quantitatively labeled with amino-derivative of fluorescein and the average amount of PMAT molecules per single nanocrystal was determined using optical absorption of the dye in the visible spectral range. The average amount of PMAT molecules grows linearly with the surface area of all studied nanocrystals. However, the surface density of the monomer units increases nonlinearly with the surface area, because of the increased competition between PMAT molecules for Zn-hexanethiol surface binding sites. The average value of zeta potential (ζ = -35 mV) was found to be independent of the size, shape, and chemical composition of nanocrystals at fixed buffer parameters (carbonate-bicarbonate buffer, pH 9.5 and 5 mM ionic strength). This finding is expected to be useful for the determination of the surface density of remaining carboxyl groups in PMAT-encapsulated nanocrystals.

Molecular beacons with JOE dye: Influence of linker and 3' couple quencher.

Molecular beacons carrying JOE dye (4',5'-dichloro-2',7'-dimethoxy-6-carboxyfluorescein) on a rigid or flexible linker and one or two BHQ1 quenchers have been prepared and tested in real-time PCR using Fusarium avenaceum elongation factor 1α DNA template. The probes were different in their structures (loop size and stem length), linkers for dye attachment (6-aminohexanol or trans-4-aminocyclohexanol), quencher composition (single and double BHQ1) to elucidate the influence of all these features. Fluorogenic properties of the probes were studied and compared to those of FAM (fluorescein)-based probes. All the factors - stem length, JOE vs FAM, rigid vs flexible linker, single vs double quencher - appeared to play a considerable role in the probe's fluorescent properties and determine the usability of the probe at two different temperatures of fluorescence detection (55°Ð¡ and 64°Ð¡).

Design of molecular beacons: 3' couple quenchers improve fluorogenic properties of a probe in real-time PCR assay.

Convenient preparation of fluorogenic hairpin DNA probes (molecular beacons) carrying a pair of FAM fluorophores (located close to 5'-terminus of the probe) or a pair of BHQ1 quenchers on 3'-terminus (with (BHQ1)2 or BHQ1-BHQ1 composition) is reported. These probes were used for the first time in a real-time PCR assay and showed considerable improvements in fluorogenic properties (the total fluorescence increase or signal-to-background ratio) in assay conditions vs. conventional one-FAM-one-BHQ1 molecular beacon probes as well as vs. hydrolyzable one-FAM-one-BHQ1 TaqMan probes. At the same time, such multiple modifications of the probe do not influence its Cq (a fractional PCR cycle used for quantification). The probe MB14 containing a BHQ1-BHQ1 pair showed a PCR fluorescence/background value of 9.6 which is more than two times higher than that of a regular probe MB2 (4.6). This study demonstrates prospects for the design of highly fluorogenic molecular beacon probes suitable for quantitative real-time PCR and for other potential applications (e.g. intracellular RNA detection and SNP/mutation analysis).

4',5'-Dichloro-2',7'-dimethoxy-5(6)-carboxyfluorescein (JOE): synthesis and spectral properties of oligonucleotide conjugates.

A convenient procedure for the preparation of the fluorescent dye 4',5'-dichloro-2',7'-dimethoxy-5(6)-carboxyfluorescein (JOE) is reported; the overall yield achieved starting from isovanillin is 10 times higher (40% vs 4%) compared to the known procedure. Isomers (5- and 6-) are easily chromatographically separable as pentafluorophenyl esters of 3',6'-O-bis(cyclohexylcarbonyl) derivatives. Four non-nucleoside JOE phosphoramidites based on 5- and 6-isomers and flexible 6-aminohexanol (AH) or rigid 4-trans-aminocyclohexanol (ACH) linkers have been prepared and used for oligonucleotide labeling. Spectral and photophysical properties of 5'-JOE-modified oligonucleotides have been studied. Fluorescence quantum yield of the dye correlates with the nature of the linker (rigid vs flexible) and with the presence of dG nucleosides in close proximity to a JOE residue.

Two-dye and one- or two-quencher DNA probes for real-time PCR assay: synthesis and comparison with a TaqMan™ probe.

A typical TaqMan™ real-time PCR probe contains a 5'-fluorescent dye and a 3'-quencher. In the course of the amplification, the probe is degraded starting from the 5'-end, thus releasing fluorescent dye. Some fluorophores (including fluorescein) are known to be prone to self-quenching when located near each other. This work is aimed at studying dye-dye and dye-quencher interactions in multiply modified DNA probes. Twenty-one fluorogenic probes containing one and two fluoresceins (FAM), or a FAM-JOE pair, and one or two BHQ1 quenchers were synthesized using non-nucleoside reagents and "click chemistry" post-modification on solid phase and in solution. The probes were tested in real-time PCR using an ~300-bp-long natural DNA fragment as a template. The structural prerequisites for lowering the probe background fluorescence and increasing the end-plateau fluorescence intensity were evaluated and discussed.

Small-Angle X-ray Scattering (SAXS) Measurements of APOBEC3G Provide Structural Basis for Binding of Single-Stranded DNA and Processivity.

APOBEC3 enzymes are polynucleotide deaminases, converting cytosine to uracil on single-stranded DNA (ssDNA) and RNA as part of the innate immune response against viruses and retrotransposons. APOBEC3G is a two-domain protein that restricts HIV. Although X-ray single-crystal structures of individual catalytic domains of APOBEC3G with ssDNA as well as full-length APOBEC3G have been solved recently, there is little structural information available about ssDNA interaction with the full-length APOBEC3G or any other two-domain APOBEC3. Here, we investigated the solution-state structures of full-length APOBEC3G with and without a 40-mer modified ssDNA by small-angle X-ray scattering (SAXS), using size-exclusion chromatography (SEC) immediately prior to irradiation to effect partial separation of multi-component mixtures. To prevent cytosine deamination, the target 2'-deoxycytidine embedded in 40-mer ssDNA was replaced by 2'-deoxyzebularine, which is known to inhibit APOBEC3A, APOBEC3B and APOBEC3G when incorporated into short ssDNA oligomers. Full-length APOBEC3G without ssDNA comprised multiple multimeric species, of which tetramer was the most scattering species. The structure of the tetramer was elucidated. Dimeric interfaces significantly occlude the DNA-binding interface, whereas the tetrameric interface does not. This explains why dimers completely disappeared, and monomeric protein species became dominant, when ssDNA was added. Data analysis of the monomeric species revealed a full-length APOBEC3G-ssDNA complex that gives insight into the observed "jumping" behavior revealed in studies of enzyme processivity. This solution-state SAXS study provides the first structural model of ssDNA binding both domains of APOBEC3G and provides data to guide further structural and enzymatic work on APOBEC3-ssDNA complexes.

Small-Angle X-ray Scattering Models of APOBEC3B Catalytic Domain in a Complex with a Single-Stranded DNA Inhibitor.

In normal cells APOBEC3 (A3A-A3H) enzymes as part of the innate immune system deaminate cytosine to uracil on single-stranded DNA (ssDNA) to scramble DNA in order to give protection against a range of exogenous retroviruses, DNA-based parasites, and endogenous retroelements. However, some viruses and cancer cells use these enzymes, especially A3A and A3B, to escape the adaptive immune response and thereby lead to the evolution of drug resistance. We have synthesized first-in-class inhibitors featuring modified ssDNA. We present models based on small-angle X-ray scattering (SAXS) data that (1) confirm that the mode of binding of inhibitor to an active A3B C-terminal domain construct in the solution state is the same as the mode of binding substrate to inactive mutants of A3A and A3B revealed in X-ray crystal structures and (2) give insight into the disulfide-linked inactive dimer formed under the oxidizing conditions of purification.

Practical synthesis of isomerically pure 5- and 6-carboxytetramethylrhodamines, useful dyes for DNA probes.

Simple and scalable synthesis of 5- and 6-carboxytetramethylrhodamines (TAMRAs) is reported. Acylation of 3-dimethylaminophenol with 1,2,4-benzenetricarboxylic anhydride afforded a mixture of 4-dimethylamino-2-hydroxy-2',4'(5')-dicarboxybenzophenones, which can be easily separated into individual compounds upon recrystallization from methanol and acetic acid. Individual benzophenones were reacted with 3-dimethylaminophenol to give 5- or 6-carboxytetramethylrhodamines. The dyes were converted into hydroxyprolinol-based phosphoramidite reagents suitable for oligonucleotide synthesis. 5- and 6-TAMRA isomers on oligonucleotides showed similar absorption and emission spectra. Fluorescence quantum yield of the dyes correlates with the presence of dG nucleosides in the adjacent region of oligonucleotide sequence. Several energy transfer primers containing on their 5'-termini (6-FAM)dT(n)(6-TAMRA) dye system (n = 0, 2, 4, 6, 8, 10, 12, 14) were prepared, and their spectral properties were studied.

Novel homo Yin-Yang probes improve sensitivity in RT-qPCR detection of low copy HIV RNA.

Nucleic acids labeled with a fluorophore/quencher pair are widely used as probes in biomedical research and molecular diagnostics. Here we synthesized novel DNA molecular beacons double labeled with the identical dyes (R6G, ROX and Cy5) at 5'- and 3'-end and studied their photo physical properties. We demonstrated that fluorescence quenching by formation of the homo dimer exciton in such molecular beacons allows using them in homogeneous assays. Further, we developed and evaluated homo Yin-Yang DNA probes labeled with identical dyes and used them for detection of low copy HIV RNA by RT-qPCR. They demonstrated improved sensitivity (LLQ: 10 vs 30 copies mL-1) in comparison to commercially available Abbott RealTime HIV-1 kit based on VIC-BHQ dyes both for model mixtures (naive human plasma with added deactivated HIV-1 virus) and for preliminarily confirmed 36 clinical samples (4 vs 1 positive ones for low-copy samples).

5-arylethynyl-2'-deoxyuridines: energy transfer and SNP-detection.

Energy transfer between different fluorescent 5-alkynyl-2' -deoxyuridines in complementary and mismatched duplexes was studied.

Reagents for the selective immobilization of oligonucleotides on solid supports.

New reagents (CPGs and phosphoramidites) for automatic solid phase synthesis of modified oligonucleotides were designed. Three oligonucleotides carrying fluorescent label at the 5'-terminus and an anchor group at the 3'-terminus were prepared and their immobilization in orthogonal conditions on solid supports was studied.

Azide phosphoramidite in direct synthesis of azide-modified oligonucleotides.

Azide and phosphoramidite functions were found to be compatible within one molecule and stable for months in solution kept frozen at -20 °C. An azide-carrying phosphoramidite was used for direct introduction of multiple azide modifications into synthetic oligonucleotides. A series of azide-containing oligonucleotides were modified further using click reactions with alkynes.

Non-nucleoside phosphoramidites of xanthene dyes (FAM, JOE, and TAMRA) for oligonucleotide labeling.

This unit describes the preparation of 5- and 6-carboxy derivatives of the xanthene fluorescent dyes fluorescein (FAM), 4',5'-dichloro-2',7'-dimethoxy-fluorescein (JOE), and tetramethylrhodamine (TAMRA) as individual isomers, and their conversion to non-nucleoside phosphoramidite reagents suitable for oligonucleotide labeling. The use of a cyclohexylcarbonyl (Chc) protecting group for blocking of phenolic hydroxyls facilitates the chromatographic separation of isomers of carboxy-FAM and carboxy-JOE as pentafluorophenyl esters. Acylation of 3-dimethylaminophenol with 1,2,4-benzenetricarboxylic anhydride gave a mixture of 4-dimethylamino-2-hydroxy-2',4'(5')-dicarboxybenzophenones, easily separable into individual compounds upon fractional crystallization. Individual isomeric benzophenones are precursors of 5- or 6-carboxytetramethylrhodamines. The dyes were converted into 6-aminohexanol- (JOE), 4-trans-aminocyclohexanol- (FAM and JOE), and hydroxyprolinol-based (TAMRA) phosphoramidite reagents.