Investigating the interactions of G-quadruplexes with transcription and chromatin structure

Four stranded G-quadruplexes (G4s) are non-canonical nucleic acid secondary structures that are primarily found in nucleosome-depleted promoters and mark sites of elevated transcription within the human genome. Although numerous studies have indicated an association between G4 formation and active transcription, the causal nature of this relationship remains unknown. The work in this thesis first addresses whether promoter G4s form as a result of transcription or whether G4 formation enhances transcription. In addition, this thesis queries the relationship between the folding of G4s and chromatin accessibility at promoters. The dependence of promoter G4 formation on transcription was tested following Inhibition of transcription elongation and initiation by 5,6-dichlorobenzimidazole 1-ß-D-ribofuranoside and triptolide respectively. Using G4 ChIP-seq, it was found that promoter G4 formation was not diminished upon transcriptional inhibition, refuting the dogma that folding of promoter G4s depends on transcriptional activity. Since endogenous G4s form primarily within sites of accessible chromatin, the impact of rapid chromatin compaction on promoter G4 folding was then assessed. Chromatin compaction was found to cause a depletion of some promoter G4s (~20%), which accompanied a reduction in RNA polymerase II (RNA Pol II) loading. This indicates that chromatin accessibility, as opposed to transcriptional activity, is a determinant of G4 formation. To assess whether G4s function to promote transcription, the small molecule ligand pyPDS was used to stabilise G4s in compacted chromatin. This was found to diminish the loss of G4s and mitigated loss of RNA Pol II at promoters upon chromatin compaction, indicating a role of G4s in promoting polymerase occupancy. Overall, these results show that G4 folding within promoters is fostered by the establishment of accessible chromatin and functions to enhance transcription by promoting Pol II loading. This builds on earlier work showing that presence of promoter G4s accompanies increased gene expression and provides evidence that the G4 structure itself may be sufficient to drive transcription by recruiting key components of the transcriptional machinery.This project is supported by Cancer Research UK core and programme award funding (C9545/A19836; C9681/A29214)

Promoter G-quadruplex folding precedes transcription and is controlled by chromatin

Funder: Herchel Smith FundsAbstract: Background: Four-stranded G-quadruplexes (G4s) are DNA secondary structures in the human genome that are primarily found in active promoters associated with elevated transcription. Here, we explore the relationship between the folding of promoter G4s, transcription and chromatin state. Results: Transcriptional inhibition by DRB or by triptolide reveals that promoter G4 formation, as assessed by G4 ChIP-seq, does not depend on transcriptional activity. We then show that chromatin compaction can lead to loss of promoter G4s and is accompanied by a corresponding loss of RNA polymerase II (Pol II), thus establishing a link between G4 formation and chromatin accessibility. Furthermore, pre-treatment of cells with a G4-stabilising ligand mitigates the loss of Pol II at promoters induced by chromatin compaction. Conclusions: Overall, our findings show that G4 folding is coupled to the establishment of accessible chromatin and does not require active transcription

Single-molecule visualization of DNA G-quadruplex formation in live cells.

Substantial evidence now exists to support that formation of DNA G-quadruplexes (G4s) is coupled to altered gene expression. However, approaches that allow us to probe G4s in living cells without perturbing their folding dynamics are required to understand their biological roles in greater detail. Herein, we report a G4-specific fluorescent probe (SiR-PyPDS) that enables single-molecule and real-time detection of individual G4 structures in living cells. Live-cell single-molecule fluorescence imaging of G4s was carried out under conditions that use low concentrations of SiR-PyPDS (20 nM) to provide informative measurements representative of the population of G4s in living cells, without globally perturbing G4 formation and dynamics. Single-molecule fluorescence imaging and time-dependent chemical trapping of unfolded G4s in living cells reveal that G4s fluctuate between folded and unfolded states. We also demonstrate that G4 formation in live cells is cell-cycle-dependent and disrupted by chemical inhibition of transcription and replication. Our observations provide robust evidence in support of dynamic G4 formation in living cells.Supported by programme grant funding from Cancer Research UK (C9681/A18618, S.B.) core funding from Cancer Research UK (C14303/A17197, S.B.), a Royal Society University Research Fellowship (UF120277 to S.F.L.), Research Professorship (RP150066 to D.K.), a EPSRC (EP/L027631/1 to D.K.) and a BBSRC David Phillips Fellowship (BB/R011605/1 to M.D.A

METHOD AND FINITE ELEMENT ANALYSIS FOR THE COMPRESSION INSTABILITY ULTIMATE LOAD OF A Z-SHAPED STIFFENED-WELED PLATE

Object of study was the large Z shaped stiffened-welded plate,combined with theoretical analysis and finite element simulation of Abaqus researched the compression instability ultimate load,to provide theory and simulation for the application of large welded plate,save the test cost and improve efficiency. The theory studied the relationship between effective width and radius of inertia,calculation of the average load as skin with truss had different material. In the simulation of analysis,ultimate load was determined by extracting the extreme value of the load proportion factor（ LPF）. For improving the accuracy of the simulation,according to the strength of the welding seam with the cloud image judged the welding sequence,studied on the variation of ultimate load in welding,to determine the limit load range. Theoretical and the simulation value compared with the experiment. Influence of the elastic modulus of material on the overall ultimate load was analyzed,by the result of the analysis,the modulus of the long truss material is more conductive to improve the ultimate load

Assessing and screening for T-cell epitopes from Mycobacterium tuberculosis RD2 proteins for the diagnosis of active tuberculosis

ABSTRACT The Region of D eletion 2 (RD2) of Mycobacterium tuberculosis encodes reserved antigens that contribute to bacterial virulence. Among these antigens, Rv1983, Rv1986, Rv1987, and Rv1989c have been shown to be immunodominant in infected cattle; however, their diagnostic utility has not been evaluated in humans.In this study, we screened 87 overlapping synthetic peptides encoded by five RD2 proteins for diagnosing tuberculosis epitopes in 50 active tuberculosis (TB) cases, 31 non-tuberculosis patients and 36 healthy individuals. A pool of promising epitopes was then assessed for their diagnostic value in 233 suspected TB patients using a whole blood IFN-γ release assay.Only 10 peptides were recognized by more than 10% of active tuberculosis patients. The IFN-γ release responses to Rv1986-P9, P15, P16, Rv1988-P4, P11, and Rv1987-P11 were significantly higher in the active TB group than in the control groups (p < 0.05). The whole blood IFN-γ release assay based on these epitopes yielded a sensitivity of 51% and a specificity of 85% in diagnosing active tuberculosis, and the corresponding results using the T-SPOT.TB assay were 76% and 75%, respectively.In conclusion, these results suggest that the six epitopes from the RD2 of M. tuberculosis have potential diagnostic value in TB

Dimerization of α-Conotoxins as a strategy to enhance the inhibition of the human α7 and α9α10 nicotinic acetylcholine receptors

The affinity of α-conotoxins, a class of nicotinic acetylcholine receptor (nAChR) peptide inhibitors, can be enhanced by dendrimerization. It has been hypothesized that this improvement arose from simultaneous binding of the α-conotoxins to several spatially adjacent sites. We here engineered several α-conotoxin dimers using a linker length compatible between neighboring binding sites on the same receptor. Remarkably, the dimer of α-conotoxin PeIA compared to the monomer displayed an increase in potency by 11-fold (IC50 = 1.9 nM) for the human α9α10 nAChR. The dimerization of α-conotoxin RgIA# resulted in a dual inhibitor that targets both α9α10 and α7 nAChR subtypes with an IC50 = ∼50 nM. The RgIA# dimer is therapeutically interesting because it is the first dual inhibitor that potently and selectively inhibits these two nAChR subtypes, which are both involved in the etiology of several cancers. We propose that the dimerization of α-conotoxins is a simpler and efficient alternative strategy to dendrimers for enhancing the activity of α-conotoxins

Promoter G-quadruplex folding precedes transcription and is controlled by chromatin

Funder: Herchel Smith FundsAbstract: Background: Four-stranded G-quadruplexes (G4s) are DNA secondary structures in the human genome that are primarily found in active promoters associated with elevated transcription. Here, we explore the relationship between the folding of promoter G4s, transcription and chromatin state. Results: Transcriptional inhibition by DRB or by triptolide reveals that promoter G4 formation, as assessed by G4 ChIP-seq, does not depend on transcriptional activity. We then show that chromatin compaction can lead to loss of promoter G4s and is accompanied by a corresponding loss of RNA polymerase II (Pol II), thus establishing a link between G4 formation and chromatin accessibility. Furthermore, pre-treatment of cells with a G4-stabilising ligand mitigates the loss of Pol II at promoters induced by chromatin compaction. Conclusions: Overall, our findings show that G4 folding is coupled to the establishment of accessible chromatin and does not require active transcription

Mechanism of Action and Structure-Activity Relationship of α-Conotoxin Mr1.1 at the Human α9α10 Nicotinic Acetylcholine Receptor

α-Conotoxins (α-CTxs) can selectively target nicotinic acetylcholine receptors (nAChRs) and are important drug leads for the treatment of cancer, chronic pain, and neuralgia. Here, we chemically synthesized a formerly defined rat α7 nAChR targeting α-CTx Mr1.1 and evaluated its activity at human nAChRs. Mr1.1 was most potent at the human (h) α9α10 nAChR with a half-maximal inhibitory concentration (IC50) of 92.0 nM. Molecular dynamic simulations suggested that Mr1.1 favorably binds at the α10(+)α9(-) and α9(+)α9(-) sites via hydrogen bonds and salt bridges, stabilizing the channel in a closed conformation. Although Mr1.1 and another antagonist, α-CTx Vc1.1 share high sequence similarity and disulfide-bond framework, Mr1.1 has distinct orientations at hα9α10. Based on the Mr1.1-hα9α10 model, analogues were generated, and the more potent Mr1.1[S4Dap], antagonized hα9α10 with an IC50of 4.0 nM. Furthermore, Mr1.1[S4Dap] displayed analgesic activity in the rat chronic constriction injury (CCI) pain model and therefore presents a promising drug candidate

Transverse flow distributions in ice‐covered channels

Abstract Traditional discharge measurements in ice‐covered river channels are time‐consuming and costly, thus limiting the elucidation of flow patterns during winter. This study proposes an analytical model for quantifying the transverse distributions of the unit‐width discharge and the depth‐averaged streamwise velocity in ice‐covered channels by extending an existing stream‐tube method to include the effects of cover and bed roughness and cross‐sectional geometry. A new set of equations are obtained for this extended method. An empirical coefficient α is included to lump the effects of the shape and friction factors of the ice cover and the channel bed. The model is compared with flume experiments and field data for fully and partially ice‐covered flows to examine the variation of the coefficient α

Rational Design of Potent α-Conotoxin PeIA Analogues with Non-Natural Amino Acids for the Inhibition of Human α9α10 Nicotinic Acetylcholine Receptors

α-Conotoxins (α-CTxs) are structurally related peptides that antagonize nicotinic acetylcholine receptors (nAChRs), which may serve as new alternatives to opioid-based treatment for pain-related conditions. The non-natural amino acid analogues of α-CTxs have been demonstrated with improved potency compared to the native peptide. In this study, we chemically synthesized Dab/Dap-substituted analogues of α-CTx PeIA and evaluated their activity at heterologously expressed human α9α10 nAChRs. PeIA[S4Dap, S9Dap] had the most potent half-maximal inhibitory concentration (IC50) of 0.93 nM. Molecular dynamic simulations suggested that the side chain amino group of Dap4 formed additional hydrogen bonds with S168 and D169 of the receptor and Dap9 formed an extra hydrogen bond interaction with Q34, which is distinctive to PeIA. Overall, our findings provide new insights into further development of more potent analogues of α-CTxs, and PeIA[S4Dap, S9Dap] has potential as a drug candidate for the treatment of chronic neuropathic pain