Journal Articles
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Item Controlling Topology of a Telomeric G-quadruplex DNA With a Chemical Cross-link(Wiley-VCH GmbH, Germany, 2025-06-18) Chilton B; Edwards PJB; Jameson GB; Hale TK; Filichev VVDNA G-quadruplexes (G4s) are noncanonical structures formed in guanine-rich sequences. Within the human genome, they are nonrandomly distributed and influence DNA replication, gene expression, and genome maintenance. Numerous proteins involved in these processes have been identified as G4-binding proteins. However, the interaction of proteins with G4s in the context of double-stranded DNA in vitro has been difficult to study due to the transient nature of G4s in the presence of complementary DNA. To overcome this challenge, introducing internal covalent cross-links between distant nucleotides within the DNA sequence may promote pre-folding of G4 structures, thereby shifting the thermodynamic equilibrium toward G4-formation. We used a Cu(I)-catalyzed azide–alkyne cycloaddition to create a cross-link between 2′-O-propargylguanosine and N6-azidoethyl-2′-deoxyadenosine in the DNA telomeric sequence (TAG3T)2. A cross-link between G3 and A8 reinforced the parallel G4 topology that was stable in the presence of complementary DNA. Moreover, even in the presence of its complementary strand, this cross-linked G4 recruited the parent native DNA (TAG3T)2 to form a hybrid G4. These results suggest that cross-linking provides a useful tool for stabilizing noncanonical DNA structures in the presence of complementary strands, enabling their study within the context of genomic DNA.Item Inverted strand polarity yields thermodynamically stable G-quadruplexes and prevents duplex formation within extended DNA.(The Royal Society of Chemistry, 2024-08-27) Chilton B; Roach RJ; Edwards PJB; Jameson GB; Hale TK; Filichev VVDNA G-quadruplexes (G4) formed in guanine-rich sequences play a key role in genome function and maintenance, interacting with multiple proteins. However, structural and functional studies of G4s within duplex DNA have been challenging because of the transient nature of G4s and thermodynamic preference of G-rich DNA to form duplexes with their complementary strand rather than G4s. To overcome these challenges, we have incorporated native nucleotides in G-rich sequences using commercially available inverted 3'-O-DMT-5'-O-phosphoramidites of native nucleosides, to give 3'-3' and 5'-5' linkages in the centre of the G-tract. Using circular dichroism and 1H nuclear magnetic resonance spectroscopies and native gel electrophoresis, we demonstrate that these polarity-inverted DNA sequences containing four telomeric repeats form G4s of parallel topology with one lateral or diagonal loop across the face of the quadruplex and two propeller loops across the edges of the quadruplex. These G4s were stable even in the presence of complementary C-rich DNA. As an example, G4 assemblies of inverted polarity were shown to bind to the hinge region of Heterochromatin Protein 1α (HP1α), a known G4-interacting domain. As such, internal polarity inversions in DNA provide a useful tool to control G4 topology while also disrupting the formation of other secondary structures, particularly the canonical duplex.Item Structure-guided inhibition of the cancer DNA-mutating enzyme APOBEC3A(Springer Nature Limited, 2023-10-11) Harjes S; Kurup HM; Rieffer AE; Bayarjargal M; Filichetva J; Su Y; Hale TK; Filichev VV; Harjes E; Harris RS; Jameson GBThe normally antiviral enzyme APOBEC3A 1-4 is an endogenous mutagen in many different human cancers 5-7 , where it becomes hijacked to fuel tumor evolvability. APOBEC3A’s single-stranded DNA C-to-U editing activity 1-8 results in multiple mutagenic outcomes including signature single-base substitution mutations (isolated and clustered), DNA breakage, and larger-scale chromosomal aberrations 5-7 . Transgenic expression in mice demonstrates its tumorigenic potential. APOBEC3A inhibitors may therefore comprise a novel class of anti-cancer agents that work by blocking mutagenesis, preventing tumor evolvability, and lessening detrimental outcomes such as drug resistance and metastasis. Here we reveal the structural basis of competitive inhibition of wildtype APOBEC3A by hairpin DNA bearing 2’-deoxy-5-fluorozebularine in place of the cytidine in the TC recognition motif that is part of a three-nucleotide loop. The nuclease-resistant phosphorothioated derivatives of these inhibitors maintain nanomolar in vitro potency against APOBEC3A, localize to the cell nucleus, and block APOBEC3A activity in human cells. These results combine to suggest roles for these inhibitors to study A3A activity in living cells, potentially as conjuvants, leading toward next-generation, combinatorial anti-mutator and anti-cancer therapies.Item Synthesis of 1,4-azaphosphinine nucleosides and evaluation as inhibitors of human cytidine deaminase and APOBEC3A.(Beilstein-Institut, 2024-05-15) Kvach MV; Harjes S; Kurup HM; Jameson GB; Harjes E; Filichev VV; Allen KNNucleoside 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.Item Structure-guided inhibition of the cancer DNA-mutating enzyme APOBEC3A.(Springer Nature Limited, 2023-10-11) Harjes S; Kurup HM; Rieffer AE; Bayarjargal M; Filitcheva J; Su Y; Hale TK; Filichev VV; Harjes E; Harris RS; Jameson GBThe normally antiviral enzyme APOBEC3A is an endogenous mutagen in human cancer. Its single-stranded DNA C-to-U editing activity results in multiple mutagenic outcomes including signature single-base substitution mutations (isolated and clustered), DNA breakage, and larger-scale chromosomal aberrations. APOBEC3A inhibitors may therefore comprise a unique class of anti-cancer agents that work by blocking mutagenesis, slowing tumor evolvability, and preventing detrimental outcomes such as drug resistance and metastasis. Here we reveal the structural basis of competitive inhibition of wildtype APOBEC3A by hairpin DNA bearing 2'-deoxy-5-fluorozebularine in place of the cytidine in the TC substrate motif that is part of a 3-nucleotide loop. In addition, the structural basis of APOBEC3A's preference for YTCD motifs (Y = T, C; D = A, G, T) is explained. The nuclease-resistant phosphorothioated derivatives of these inhibitors have nanomolar potency in vitro and block APOBEC3A activity in human cells. These inhibitors may be useful probes for studying APOBEC3A activity in cellular systems and leading toward, potentially as conjuvants, next-generation, combinatorial anti-mutator and anti-cancer therapies.Item Seven-membered ring nucleobases as inhibitors of human cytidine deaminase and APOBEC3A.(Royal Society of Chemistry, 2023-06-21) Kurup HM; Kvach MV; Harjes S; Jameson GB; Harjes E; Filichev VVThe 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.Item [Fe(µ2-OH)6]3- Linked Fe3O Triads: Mössbauer Evidence for Trigonal µ3-O2- or µ3-OH- Groups in Bridged versus Unbridged Complexes.(MDPI (Basel, Switzerland), 2024-07-07) De Silva DNT; Dais TN; Jameson GB; Davies CG; Jameson GNL; Plieger PG; Niclós-Gutiérrez J; Barceló-Oliver MThe syntheses, coordination chemistry, and Mössbauer spectroscopy of hepta-iron(III) complexes using derivatised salicylaldoxime ligands from two categories; namely, 'single-headed' (H2L) and 'double-headed' (H4L) salicylaldoximes are described. All compounds presented here share a [Fe3-µ3-O] core in which the iron(III) ions are µ3-hydroxo-bridged in the complex C1 and µ3-oxo-bridged in C2 and C3. Each compound consists of 2 × [Fe3-µ3-O] triads that are linked via a central [Fe(µ2-OH)6]3- ion. In addition to the charge balance and microanalytical evidence, Mössbauer measurements support the fact that the triads in C1 are µ3-OH bridged and are µ3-O bridged in C2 and C3.Item Assessment of Various Food Proteins as Structural Materials for Delivery of Hydrophobic Polyphenols Using a Novel Co-Precipitation Method(MDPI (Basel, Switzerland), 2023-04-19) Rashidinejad A; Nieuwkoop M; Singh H; Jameson GB; Papetti AIn this study, sodium caseinate (NaCas), soy protein isolate (SPI), and whey protein isolate (WPI) were used as structural materials for the delivery of rutin, naringenin, curcumin, hesperidin, and catechin. For each polyphenol, the protein solution was brought to alkaline pH, and then the polyphenol and trehalose (as a cryo-protectant) were added. The mixtures were later acidified, and the co-precipitated products were lyophilized. Regardless of the type of protein used, the co-precipitation method exhibited relatively high entrapment efficiency and loading capacity for all five polyphenols. Several structural changes were seen in the scanning electron micrographs of all polyphenol-protein co-precipitates. This included a significant decrease in the crystallinity of the polyphenols, which was confirmed by X-ray diffraction analysis, where amorphous structures of rutin, naringenin, curcumin, hesperidin, and catechin were revealed after the treatment. Both the dispersibility and solubility of the lyophilized powders in water were improved dramatically (in some cases, >10-fold) after the treatment, with further improvements observed in these properties for the powders containing trehalose. Depending on the chemical structure and hydrophobicity of the tested polyphenols, there were differences observed in the degree and extent of the effect of the protein on different properties of the polyphenols. Overall, the findings of this study demonstrated that NaCas, WPI, and SPI can be used for the development of an efficient delivery system for hydrophobic polyphenols, which in turn can be incorporated into various functional foods or used as supplements in the nutraceutical industry.Item The LONELY GUY gene family: from mosses to wheat, the key to the formation of active cytokinins in plants(John Wiley and Sons Ltd on behalf of the Society for Experimental Biology and The Association of Applied Biologists, 2022-04-07) Chen L; Jameson GB; Guo Y; Song J; Jameson PELONELY GUY (LOG) was first identified in a screen of rice mutants with defects in meristem maintenance. In plants, LOG codes for cytokinin riboside 5'-monophosphate phosphoribohydrolase, which converts inactive cytokinin nucleotides directly to the active free bases. Many enzymes with the PGGxGTxxE motif have been misannotated as lysine decarboxylases; conversely not all enzymes containing this motif are cytokinin-specific LOGs. As LOG mutants clearly impact yield in rice, we investigated the LOG gene family in bread wheat. By interrogating the wheat (Triticum aestivum) genome database, we show that wheat has multiple LOGs. The close alignment of TaLOG1, TaLOG2 and TaLOG6 with the X-ray structures of two functional Arabidopsis thaliana LOGs allows us to infer that the wheat LOGs 1-11 are functional LOGs. Using RNA-seq data sets, we assessed TaLOG expression across 70 tissue types, their responses to various stressors, the pattern of cis-regulatory elements (CREs) and intron/exon patterns. TaLOG gene family members are expressed variously across tissue types. When the TaLOG CREs are compared with those of the cytokinin dehydrogenases (CKX) and glucosyltransferases (CGT), there is close alignment of CREs between TaLOGs and TaCKXs reflecting the key role of CKX in maintaining cytokinin homeostasis. However, we suggest that the main homeostatic mechanism controlling cytokinin levels in response to biotic and abiotic challenge resides in the CGTs, rather than LOG or CKX. However, LOG transgenics and identified mutants in rice variously impact yield, providing interesting avenues for investigation in wheat.Item The Effect of pH and Sodium Caseinate on the Aqueous Solubility, Stability, and Crystallinity of Rutin towards Concentrated Colloidally Stable Particles for the Incorporation into Functional Foods(MDPI (Basel, Switzerland), 2022-01-14) Rashidinejad A; Jameson GB; Singh H; Papetti APoor water solubility and low bioavailability of hydrophobic flavonoids such as rutin remain as substantial challenges to their oral delivery via functional foods. In this study, the effect of pH and the addition of a protein (sodium caseinate; NaCas) on the aqueous solubility and stability of rutin was studied, from which an efficient delivery system for the incorporation of rutin into functional food products was developed. The aqueous solubility, chemical stability, crystallinity, and morphology of rutin (0.1-5% w/v) under various pH (1-11) and protein concentrations (0.2-8% w/v) were studied. To manufacture the concentrated colloidally stable rutin-NaCas particles, rutin was dissolved and deprotonated in a NaCas solution at alkaline pH before its subsequent neutralisation at pH 7. The excess water was removed using ultrafiltration to improve the loading capacity. Rutin showed the highest solubility at pH 11, while the addition of NaCas resulted in the improvement of both solubility and chemical stability. Critically, to achieve particles with colloidal stability, the NaCas:rutin ratio (w/w) had to be greater than 2.5 and 40 respectively for the lowest (0.2% w/v) and highest (4 to 8% w/v) concentrations of NaCas. The rutin-NaCas particles in the concentrated formulations were physically stable, with a size in the range of 185 to 230 nm and zeta potential of -36.8 to -38.1 mV, depending on the NaCas:rutin ratio. Encapsulation efficiency and loading capacity of rutin in different systems were 76% to 83% and 2% to 22%, respectively. The concentrated formulation containing 5% w/v NaCas and 2% w/v rutin was chosen as the most efficient delivery system due to the ideal protein:flavonoid ratio (2.5:1), which resulted in the highest loading capacity (22%). Taken together, the findings show that the delivery system developed in this study can be a promising method for the incorporation of a high concentration of hydrophobic flavonoids such as rutin into functional foods.