Structure, internal motions and association–dissociation kinetics of the i-motif dimer of d(5mCCTCACTCC)

At slightly acidic pH, the association of two d(5mCCTCACTCC) strands results in the formation of an i-motif dimer. Using NMR methods, we investigated the structure of [d(5mCCTCACTCC)](2), the internal motion of the base pairs stacked in the i-motif core, the dimer formation and dissociation kinetics versus pH. The excellent resolution of the (1)H and (31)P spectra provided the determination of dihedral angles, which together with a large set of distance restraints, improve substantially the definition of the sugar-phosphate backbone by comparison with previous NMR studies of i-motif structures. [d(5mCCTCACTCC)](2) is built by intercalation of two symmetrical hairpins held together by six symmetrical C•C(+) pairs and by pair T7•T7. The hairpin loops that are formed by a single residue, A5, cross the narrow grooves on the same side of the i-motif core. The base pair intercalation order is C9•C9(+)/5mC1•5mC1(+)/C8•C8(+)/C2•C2(+)/T7.T7/C6•C6(+)/C4•C4(+). The T3 bases are flipped out in the wide grooves. The core of the structure includes four long-lived pairs whose lifetimes at 15°C range from 100 s (C8•C8(+)) to 0.18 s (T7•T7). The formation rate and the lifetime of [d(5mCCTCACTCC)](2) were measured between pH 6.8 and 4.8. The dimer formation rate is three to four magnitude orders slower than that of a B-DNA duplex. It depends on pH, as it must occur for a bimolecular process involving non cooperative association of neutral and protonated residues. In the range of pH investigated, the dimer lifetime, 500 s at 0°C, pH 6.8, varies approximately as 10(−pH)

[C7GC4]4 Association into supra molecular i-motif structures

The self-associative properties of cytidine-rich oligonucleotides into symmetrical i-motif tetramers give to these oligonucleotides the capacity of forming supramolecular structures (sms) that have potential applications in the nanotechnology domain. In order to facilitate sms formation, oligonucleotides containing two cytidine stretches of unequal length (CnXCm) separated by a non-cytidine spacer were synthesized. They were designed to associate into a tetramer including an i-motif core built by intercalation of the C·C+ pairs of the longer C stretch with the two dangling non-intercalated strands of the shorter C stretch at each end. Gel filtration chromatography shows that the non-intercalated C-rich ends give to this structure the capacity of forming extremely stable sms. Using C7GC4 as a model, we find that the sms formation rate varies as the oligonucleotide concentration and increases at high temperature. Competitively with the tetramer involved in sms elongation, CnXCm oligonucleotides form i-motif dimers that compete with sms elongation. The dimer stability is strongly reduced when the pH is moved away from the cytidine pK. This results in an equilibrium shift towards the tetramer and in the acceleration of the sms formation rate. The chromatograms of the sms formed by C7GC4 indicate a broad distribution. In a 1.5 mM solution incubated at 37°C, the equilibrium distribution is centered on a molecular weight corresponding to the assembly of nine tetramers and the upper limit corresponds to 80 tetramers. The lifetime of this structure is about 4 days at 40°C, pH 4.6

The formation pathway of i-motif tetramers

The i-motif is a four-stranded structure formed by two intercalated parallel duplexes containing hemiprotonated C•C+ pairs. In order to describe the sequence of reactions by which four C-rich strands associate, we measured the formation and dissociation rates of three [TCn]4 tetramers (n = 3, 4 and 5), their dissociation constant and the reaction order for tetramer formation by NMR. We find that TCn association results in the formation of several tetramers differing by the number of intercalated C•C+ pairs. The formation rates of the fully and partially intercalated species are comparable but their lifetimes increase strongly with the number of intercalated C•C+ pairs, and for this reason the single tetramer detected at equilibrium is that with optimal intercalation. The tetramer half formation times vary as the power −2 of the oligonucleotide concentration indicating that the reaction order for i-motif formation is 3. This observation is inconsistent with a model supposing association of two preformed duplex and suggests that quadruplex formation proceeds via sequential strand association into duplex and triplex intermediate species and that triplex formation is rate limiting

NMR solution structure of the major G-quadruplex structure formed in the human BCL2 promoter region

BCL2 protein functions as an inhibitor of cell apoptosis and has been found to be aberrantly expressed in a wide range of human diseases. A highly GC-rich region upstream of the P1 promoter plays an important role in the transcriptional regulation of BCL2. Here we report the NMR solution structure of the major intramolecular G-quadruplex formed on the G-rich strand of this region in K(+) solution. This well-defined mixed parallel/antiparallel-stranded G-quadruplex structure contains three G-tetrads of mixed G-arrangements, which are connected with two lateral loops and one side loop, and four grooves of different widths. The three loops interact with the core G-tetrads in a specific way that defines and stabilizes the overall G-quadruplex structure. The loop conformations are in accord with the experimental mutation and footprinting data. The first 3-nt loop adopts a lateral loop conformation and appears to determine the overall folding of the BCL2 G-quadruplex. The third 1-nt double-chain-reversal loop defines another example of a stable parallel-stranded structural motif using the G(3)NG(3) sequence. Significantly, the distinct major BCL2 promoter G-quadruplex structure suggests that it can be specifically involved in gene modulation and can be an attractive target for pathway-specific drug design

EDUCATIONAL PROJECT ON LUNG CANCER PATIENTS UNDERGOING MOLECULAR TARGET THERAPY AT ULSS 7 PEDEMONTANA

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Structure et mouvement internes d'oligonucléotides d'ADN et ARN

PARIS-BIUSJ-Thèses (751052125) / SudocPARIS-BIUSJ-Physique recherche (751052113) / SudocSudocFranceF

Opening mechanism of G.T/U pairs in DNA and RNA duplexes: A combined study of imino proton exchange and molecular dynamics simulation

The opening pathway of wobble pairs dT and rU has been investigated in four DNA and two RNA duplexes. Using NMR spectroscopy, we measured the imino proton exchange of both G(H1) and T/U(H3), catalyzed by ammonia, tris, and OH-, and we calculated the free energy surface related to T/U opening by molecular dynamics simulations. Taken together the experimental and theoretical results, we suggest that wobble pairs open through a coupled rotation of the bases toward the major groove where exchange of both imino protons takes place with the surrounding water

Field Validation of Commercially Available Food Retailer Data in the Netherlands

The aim of this study was to validate a Dutch commercial dataset containing information on the types and locations of food retailers against field audit data. Field validation of a commercial dataset ("Locatus") was conducted in February 2019. Data on the location and classification of food retailers were collected through field audits in 152 streets from four urban and four rural neighborhoods in the Netherlands. The classification of food retailers included eight types of grocery stores (e.g., supermarkets, bakeries) and four types of food outlets (e.g., cafés, take away restaurants). The commercial dataset in the studied area listed 322 food retailers, whereas the field audit counted 315 food retailers. Overall, the commercially available data showed "good" to "excellent" agreement statistics (>0.71) with field audit data for all three levels of analysis (i.e., location, classification and both combined) and across urban as well as rural areas. The commercial dataset under study provided an accurate description of the measured food environment. Therefore, policymakers and researchers should feel confident in using this commercial dataset as a source of secondary data