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1.
Genes Chromosomes Cancer ; 62(3): 121-130, 2023 03.
Article in English | MEDLINE | ID: mdl-36326821

ABSTRACT

Tumor mutational burden (TMB), measured by exome or panel sequencing of tumor tissue or blood (bTMB), is a potential predictive biomarker for treatment benefit in patients with various cancer types receiving immunotherapy targeting checkpoint pathways. However, significant variability in TMB measurement has been observed. We developed contrived bTMB reference materials using DNA from tumor cell lines and donor-matched lymphoblastoid cell lines to support calibration and alignment across laboratories and platforms. Contrived bTMB reference materials were developed using genomic DNA from lung tumor cell lines blended into donor-matched lymphoblastoid cell lines at 0.5% and 2% tumor content, fragmented and size-selected to mirror the size profile of circulating cell-free tumor DNA with TMB scores of 7, 9, 20, and 26 mut/Mb. Variant allele frequency (VAF) and bTMB scores were assessed using PredicineATLAS and GuardantOMNI next-generation sequencing assays. DNA fragment sizes in the contrived reference samples were similar to those found within patient plasma-derived cell-free DNA, and mutational patterns aligned with those in the parental tumor lines. For the 7, 20, and 26 mut/Mb contrived reference samples with 2% tumor content, bTMB scores estimated using either assay aligned with expected scores from the parental tumor cell lines and showed good reproducibility. A bioinformatic filtration step was required to account for low-VAF artifact variants. We demonstrate the feasibility and challenges of producing and using bTMB reference standards across a range of bTMB levels, and how such standards could support the calibration and validation of bTMB platforms and help harmonization between panels and laboratories.


Subject(s)
Lung Neoplasms , Neoplasms , Humans , Reproducibility of Results , Neoplasms/genetics , Mutation , Immunotherapy , Biomarkers, Tumor/genetics , Biomarkers, Tumor/metabolism , Lung Neoplasms/genetics
2.
Proc Natl Acad Sci U S A ; 117(13): 7216-7224, 2020 03 31.
Article in English | MEDLINE | ID: mdl-32165536

ABSTRACT

Protein flexibility and disorder is emerging as a crucial modulator of chromatin structure. Histone tail disorder enables transient binding of different molecules to the nucleosomes, thereby promoting heterogeneous and dynamic internucleosome interactions and making possible recruitment of a wide-range of regulatory and remodeling proteins. On the basis of extensive multiscale modeling we reveal the importance of linker histone H1 protein disorder for chromatin hierarchical looping. Our multiscale approach bridges microsecond-long bias-exchange metadynamics molecular dynamics simulations of atomistic 211-bp nucleosomes with coarse-grained Monte Carlo simulations of 100-nucleosome systems. We show that the long C-terminal domain (CTD) of H1-a ubiquitous nucleosome-binding protein-remains disordered when bound to the nucleosome. Notably, such CTD disorder leads to an asymmetric and dynamical nucleosome conformation that promotes chromatin structural flexibility and establishes long-range hierarchical loops. Furthermore, the degree of condensation and flexibility of H1 can be fine-tuned, explaining chromosomal differences of interphase versus metaphase states that correspond to partial and hyperphosphorylated H1, respectively. This important role of H1 protein disorder in large-scale chromatin organization has a wide range of biological implications.


Subject(s)
Chromatin/physiology , DNA-Binding Proteins/physiology , Animals , Chromatin/genetics , Chromosomal Proteins, Non-Histone/physiology , DNA-Binding Proteins/metabolism , Histones/metabolism , Humans , Metaphase , Models, Molecular , Nucleic Acid Conformation , Nucleosomes/physiology , Protein Binding/physiology
3.
PLoS Comput Biol ; 17(11): e1009547, 2021 11.
Article in English | MEDLINE | ID: mdl-34748533

ABSTRACT

We present a comprehensive, experimental and theoretical study of the impact of 5-hydroxymethylation of DNA cytosine. Using molecular dynamics, biophysical experiments and NMR spectroscopy, we found that Ten-Eleven translocation (TET) dioxygenases generate an epigenetic variant with structural and physical properties similar to those of 5-methylcytosine. Experiments and simulations demonstrate that 5-methylcytosine (mC) and 5-hydroxymethylcytosine (hmC) generally lead to stiffer DNA than normal cytosine, with poorer circularization efficiencies and lower ability to form nucleosomes. In particular, we can rule out the hypothesis that hydroxymethylation reverts to unmodified cytosine physical properties, as hmC is even more rigid than mC. Thus, we do not expect dramatic changes in the chromatin structure induced by differences in physical properties between d(mCpG) and d(hmCpG). Conversely, our simulations suggest that methylated-DNA binding domains (MBDs), associated with repression activities, are sensitive to the substitution d(mCpG) ➔ d(hmCpG), while MBD3 which has a dual activation/repression activity is not sensitive to the d(mCpG) d(hmCpG) change. Overall, while gene activity changes due to cytosine methylation are the result of the combination of stiffness-related chromatin reorganization and MBD binding, those associated to 5-hydroxylation of methylcytosine could be explained by a change in the balance of repression/activation pathways related to differential MBD binding.


Subject(s)
5-Methylcytosine/analogs & derivatives , DNA Methylation , DNA/chemistry , DNA/metabolism , Epigenesis, Genetic , 5-Methylcytosine/chemistry , 5-Methylcytosine/metabolism , Binding Sites , Biophysical Phenomena , Computational Biology , DNA/genetics , Humans , Magnetic Resonance Spectroscopy , Models, Biological , Molecular Dynamics Simulation , Nucleic Acid Conformation
4.
Biochemistry ; 59(4): 379-388, 2020 02 04.
Article in English | MEDLINE | ID: mdl-31815441

ABSTRACT

It is increasingly recognized that the structures and dynamics of G-quadruplex DNA molecules are dictated by their sequences and greatly affected by environmental factors. The core guanine tetrads (G-tetrads) coordinate cations and display a strong conformational rigidity compared with that of the connecting loops. Although long loops linking the G-tetrads are typically disfavored, when present, they provide a striking illustration of the dynamics of short, single-stranded DNA regions. In addition to their role in determining the stability of the G-quadruplex state, these loops are also interesting as potential drug targets. To characterize accurately the dynamics of this DNA state, we apply here the principles of structural ensemble determination developed in the past two decades for protein molecules to DNA molecules. We thus perform extensive molecular dynamics simulations restrained with nuclear magnetic resonance residual dipolar couplings to determine a structural ensemble of the human CEB25 minisatellite G-quadruplex, which contains a connecting loop of nine nucleotides. This structural ensemble displays a wide set of arrangements for the loop and a compact, well-defined G-quadruplex core. Our results show the importance of stacking interactions in the loop and strengthen the ability of the closing base pairs to confer a large thermodynamic stability to the G-quadruplex structure.


Subject(s)
DNA/chemistry , DNA/ultrastructure , G-Quadruplexes , Guanine/chemistry , Guanine/metabolism , Humans , Magnetic Resonance Spectroscopy/methods , Molecular Dynamics Simulation , Nuclear Magnetic Resonance, Biomolecular/methods , Nucleic Acid Conformation , Thermodynamics
5.
Nat Methods ; 13(1): 55-8, 2016 Jan.
Article in English | MEDLINE | ID: mdl-26569599

ABSTRACT

We present parmbsc1, a force field for DNA atomistic simulation, which has been parameterized from high-level quantum mechanical data and tested for nearly 100 systems (representing a total simulation time of ∼ 140 µs) covering most of DNA structural space. Parmbsc1 provides high-quality results in diverse systems. Parameters and trajectories are available at http://mmb.irbbarcelona.org/ParmBSC1/.


Subject(s)
DNA/chemistry , Quantum Theory
6.
Nucleic Acids Res ; 45(7): 4217-4230, 2017 04 20.
Article in English | MEDLINE | ID: mdl-28088759

ABSTRACT

Last generation of force-fields are raising expectations on the quality of molecular dynamics (MD) simulations of DNA, as well as to the belief that theoretical models can substitute experimental ones in several cases. However these claims are based on limited benchmarks, where MD simulations have shown the ability to reproduce already existing 'experimental models', which in turn, have an unclear accuracy to represent DNA conformation in solution. In this work we explore the ability of different force-fields to predict the structure of two new B-DNA dodecamers, determined herein by means of 1H nuclear magnetic resonance (NMR). The study allowed us to check directly for experimental NMR observables on duplexes previously not solved, and also to assess the reliability of 'experimental structures'. We observed that technical details in the annealing procedures can induce non-negligible local changes in the final structures. We also found that while not all theoretical simulations are equally reliable, those obtained using last generation of AMBER force-fields (BSC1 and BSC0OL15) show predictive power in the multi-microsecond timescale and can be safely used to reproduce global structure of DNA duplexes and fine sequence-dependent details.


Subject(s)
DNA, B-Form/chemistry , Molecular Dynamics Simulation , Nuclear Magnetic Resonance, Biomolecular , Base Sequence , Crystallography, X-Ray , Nucleic Acid Conformation
7.
J Am Chem Soc ; 137(32): 10205-15, 2015 Aug 19.
Article in English | MEDLINE | ID: mdl-26192632

ABSTRACT

Histone tails and their epigenetic modifications play crucial roles in gene expression regulation by altering the architecture of chromatin. However, the structural mechanisms by which histone tails influence the interconversion between active and inactive chromatin remain unknown. Given the technical challenges in obtaining detailed experimental characterizations of the structure of chromatin, multiscale computations offer a promising alternative to model the effect of histone tails on chromatin folding. Here we combine multimicrosecond atomistic molecular dynamics simulations of dinucleosomes and histone tails in explicit solvent and ions, performed with three different state-of-the-art force fields and validated by experimental NMR measurements, with coarse-grained Monte Carlo simulations of 24-nucleosome arrays to describe the conformational landscape of histone tails, their roles in chromatin compaction, and the impact of lysine acetylation, a widespread epigenetic change, on both. We find that while the wild-type tails are highly flexible and disordered, the dramatic increase of secondary-structure order by lysine acetylation unfolds chromatin by decreasing tail availability for crucial fiber-compacting internucleosome interactions. This molecular level description of the effect of histone tails and their charge modifications on chromatin folding explains the sequence sensitivity and underscores the delicate connection between local and global structural and functional effects. Our approach also opens new avenues for multiscale processes of biomolecular complexes.


Subject(s)
Chromatin/chemistry , Chromatin/genetics , Epigenesis, Genetic , Histones/chemistry , Acetylation , Chromatin/metabolism , Histones/metabolism , Lysine/metabolism , Magnetic Resonance Spectroscopy , Molecular Dynamics Simulation , Monte Carlo Method , Nucleosomes/chemistry , Nucleosomes/metabolism
8.
J Org Chem ; 80(6): 3083-91, 2015 Mar 20.
Article in English | MEDLINE | ID: mdl-25723361

ABSTRACT

We report the synthesis, thermal stability, and RNase H substrate activity of 2'-deoxy-2',4'-difluoroarabino-modified nucleic acids. 2'-Deoxy-2',4'-difluoroarabinouridine (2,'4'-diF-araU) was prepared in a stereoselective way in six steps from 2'-deoxy-2'-fluoroarabinouridine (2'-F-araU). NMR analysis and quantum mechanical calculations at the nucleoside level reveal that introduction of 4'-fluorine introduces a strong bias toward the North conformation, despite the presence of the 2'-ßF, which generally steers the sugar pucker toward the South/East conformation. Incorporation of the novel monomer into DNA results on a neutral to slightly stabilizing thermal effect on DNA-RNA hybrids. Insertion of 2',4'-diF-araU nucleotides in the DNA strand of a DNA-RNA hybrid decreases the rate of both human and HIV reverse transcriptase-associated RNase H-mediated cleavage of the complement RNA strand compared to that for an all-DNA strand or a DNA strand containing the corresponding 2'-F-araU nucleotide units, consistent with the notion that a 4'-fluorine in 2'-F-araU switches the preferred sugar conformation from DNA-like (South/East) to RNA-like (North).


Subject(s)
Arabinose/analogs & derivatives , DNA/chemistry , DNA/chemical synthesis , RNA/chemistry , RNA/chemical synthesis , Arabinose/chemistry , Molecular Structure
9.
Angew Chem Int Ed Engl ; 54(36): 10488-91, 2015 Sep 01.
Article in English | MEDLINE | ID: mdl-26224143

ABSTRACT

The stability of DNA is highly dependent on the properties of the surrounding solvent, such as ionic strength, pH, and the presence of denaturants and osmolytes. Addition of pyridine is known to unfold DNA by replacing π-π stacking interactions between bases, stabilizing conformations in which the nucleotides are solvent exposed. We show here experimental and theoretical evidences that pyridine can change its role and in fact stabilize the DNA under acidic conditions. NMR spectroscopy and MD simulations demonstrate that the reversal in the denaturing role of pyridine is specific, and is related to its character as pseudo groove binder. The present study sheds light on the nature of DNA stability and on the relationship between DNA and solvent, with clear biotechnological implications.


Subject(s)
Acids/chemistry , DNA/chemistry , Hydrogen-Ion Concentration , Nucleic Acid Denaturation , Pyridines/chemistry , Molecular Dynamics Simulation
10.
Angew Chem Int Ed Engl ; 54(2): 467-71, 2015 Jan 07.
Article in English | MEDLINE | ID: mdl-25417598

ABSTRACT

By combining ion-mobility mass spectrometry experiments with sub-millisecond classical and ab initio molecular dynamics we fully characterized, for the first time, the dynamic ensemble of a model nucleic acid in the gas phase under electrospray ionization conditions. The studied oligonucleotide unfolds upon vaporization, loses memory of the solution structure, and explores true gas-phase conformational space. Contrary to our original expectations, the oligonucleotide shows very rich dynamics in three different timescales (multi-picosecond, nanosecond, and sub-millisecond). The shorter timescale dynamics has a quantum mechanical nature and leads to changes in the covalent structure, whereas the other two are of classical origin. Overall, this study suggests that a re-evaluation on our view of the physics of nucleic acids upon vaporization is needed.


Subject(s)
Gases/chemical synthesis , Oligonucleotides/chemistry , Molecular Structure
11.
J Am Chem Soc ; 136(8): 3075-86, 2014 Feb 26.
Article in English | MEDLINE | ID: mdl-24490755

ABSTRACT

It has been known for decades that alkylammonium ions, such as tetramethyl ammonium (TMA), alter the usual correlation between DNA GC-content and duplex stability. In some cases it is even possible for an AT-rich duplex to be more stable than a GC-rich duplex of the same length. There has been much speculation regarding the origin of this aberration in sequence-dependent DNA duplex stability, but no clear resolution. Using a combination of molecular dynamics simulations and NMR spectroscopy we demonstrate that choline (2-hydroxy-N,N,N-trimethylethanaminium) and TMA are preferentially localized in the minor groove of DNA duplexes at A·T base pairs and these same ions show less pronounced localization in the major groove compared to what has been demonstrated for alkali and alkali earth metal ions. Furthermore, free energy calculations show that single-stranded GC-rich sequences exhibit more favorable solvation by choline than single-stranded AT-rich sequences. The sequence-specific nature of choline and TMA binding provides a rationale for the enhanced stability of AT-rich sequences when alkyl-ammonium ions are used as the counterions of DNA. Our combined theoretical and experimental study provides one of the most detailed pictures to date of cations localized along DNA in the solution state, and provides insights that go beyond understanding alkyl-ammonium ion binding to DNA. In particular, because choline and TMA bind to DNA in a manner that is found to be distinct from that previously reported for Na(+), K(+), Mg(2+), and Ca(2+), our results reveal the important but underappreciated role that most other cations play in sequence-specific duplex stability.


Subject(s)
Choline/chemistry , DNA/chemistry , Nucleic Acid Conformation , Quaternary Ammonium Compounds/chemistry , GC Rich Sequence , Magnetic Resonance Spectroscopy , Molecular Dynamics Simulation
12.
PLoS Comput Biol ; 9(11): e1003354, 2013.
Article in English | MEDLINE | ID: mdl-24278005

ABSTRACT

Cytosine methylation is one of the most important epigenetic marks that regulate the process of gene expression. Here, we have examined the effect of epigenetic DNA methylation on nucleosomal stability using molecular dynamics simulations and elastic deformation models. We found that methylation of CpG steps destabilizes nucleosomes, especially when these are placed in sites where the DNA minor groove faces the histone core. The larger stiffness of methylated CpG steps is a crucial factor behind the decrease in nucleosome stability. Methylation changes the positioning and phasing of the nucleosomal DNA, altering the accessibility of DNA to regulatory proteins, and accordingly gene functionality. Our theoretical calculations highlight a simple physical-based explanation on the foundations of epigenetic signaling.


Subject(s)
DNA Methylation , Epigenesis, Genetic , Models, Genetic , Nucleosomes/genetics , Computational Biology , Cytosine/chemistry , Cytosine/metabolism , Molecular Dynamics Simulation , Thermodynamics
13.
Proc Natl Acad Sci U S A ; 108(10): 3935-40, 2011 Mar 08.
Article in English | MEDLINE | ID: mdl-21368142

ABSTRACT

Transporters of the amino acid, polyamine and organocation (APC) superfamily play essential roles in cell redox balance, cancer, and aminoacidurias. The bacterial L-arginine/agmatine antiporter, AdiC, is the main APC structural paradigm and shares the "5 + 5 inverted repeat" fold found in other families like the Na(+)-coupled neurotransmitter transporters. The available AdiC crystal structures capture two states of its transport cycle: the open-to-out apo and the outward-facing Arg(+)-bound occluded. However, the role of Arg(+) during the transition between these two states remains unknown. Here, we report the crystal structure at 3.0 Å resolution of an Arg(+)-bound AdiC mutant (N101A) in the open-to-out conformation, completing the picture of the major conformational states during the transport cycle of the 5 + 5 inverted repeat fold-transporters. The N101A structure is an intermediate state between the previous known AdiC conformations. The Arg(+)-guanidinium group in the current structure presents high mobility and delocalization, hampering substrate occlusion and resulting in a low translocation rate. Further analysis supports that proper coordination of this group with residues Asn101 and Trp293 is required to transit to the occluded state, providing the first clues on the molecular mechanism of substrate-induced fit in a 5 + 5 inverted repeat fold-transporter. The pseudosymmetry found between repeats in AdiC, and in all fold-related transporters, restraints the conformational changes, in particular the transmembrane helices rearrangements, which occur during the transport cycle. In AdiC these movements take place away from the dimer interface, explaining the independent functioning of each subunit.


Subject(s)
Amino Acids/metabolism , Carrier Proteins/metabolism , Carrier Proteins/chemistry , Crystallography, X-Ray , Models, Molecular , Protein Conformation
14.
J Am Chem Soc ; 135(14): 5344-7, 2013 Apr 10.
Article in English | MEDLINE | ID: mdl-23521511

ABSTRACT

Human telomeric DNA quadruplexes can adopt different conformations in solution. We have found that arabinose, 2'F-arabinose, and ribose substitutions stabilize the propeller parallel G-quadruplex form over competing conformers, allowing NMR structural determination of this particularly significant nucleic acid structure. 2'F-arabinose substitution provides the greatest stabilization as a result of electrostatic (F-CH---O4') and pseudo-hydrogen-bond (F---H8) stabilizing interactions. In contrast, 2'F-rG substitution provokes a dramatic destabilization of the quadruplex structure due to unfavorable electrostatic repulsion between the phosphate and the 2'-F.


Subject(s)
Arabinose/chemistry , DNA/chemistry , Fluorine/chemistry , G-Quadruplexes , Telomere/chemistry , Humans , Models, Molecular , Molecular Structure , Protein Folding
15.
Biophys J ; 103(8): 1698-705, 2012 Oct 17.
Article in English | MEDLINE | ID: mdl-23083713

ABSTRACT

Water permeability through single-file channels is affected by intrinsic factors such as their size and polarity and by external determinants like their lipid environment in the membrane. Previous computational studies revealed that the obstruction of the channel by lipid headgroups can be long-lived, in the range of nanoseconds, and that pore-length-matching membrane mimetics could speed up water permeability. To test the hypothesis of lipid-channel interactions modulating channel permeability, we designed different gramicidin A derivatives with attached acyl chains. By combining extensive molecular-dynamics simulations and single-channel water permeation measurements, we show that by tuning lipid-channel interactions, these modifications reduce the presence of lipid headgroups in the pore, which leads to a clear and selective increase in their water permeability.


Subject(s)
Gramicidin/chemistry , Molecular Dynamics Simulation , Water/chemistry , Acylation , Cell Membrane/chemistry , Cell Membrane/metabolism , Lipid Bilayers/chemistry , Lipid Bilayers/metabolism , Permeability , Water/metabolism
16.
J Am Chem Soc ; 134(15): 6596-606, 2012 Apr 18.
Article in English | MEDLINE | ID: mdl-22420643

ABSTRACT

Extensive (more than 90 microseconds) molecular dynamics simulations complemented with ion-mobility mass spectrometry experiments have been used to characterize the conformational ensemble of DNA triplexes in the gas phase. Our results suggest that the ensemble of DNA triplex structures in the gas phase is well-defined over the experimental time scale, with the three strands tightly bound, and for the most abundant charge states it samples conformations only slightly more compact than the solution structure. The degree of structural alteration is however very significant, mimicking that found in duplex and much larger than that suggested for G-quadruplexes. Our data strongly supports that the gas phase triplex maintains an excellent memory of the solution structure, well-preserved helicity, and a significant number of native contacts. Once again, a linear, flexible, and charged polymer as DNA surprises us for its ability to retain three-dimensional structure in the absence of solvent. Results argue against the generally assumed roles of the different physical interactions (solvent screening of phosphate repulsion, hydrophobic effect, and solvation of accessible polar groups) in modulating the stability of DNA structures.


Subject(s)
DNA/chemistry , Gases/chemistry , Mass Spectrometry , Molecular Dynamics Simulation , Nucleic Acid Conformation
17.
PLoS Comput Biol ; 7(3): e1001098, 2011 Mar.
Article in English | MEDLINE | ID: mdl-21390270

ABSTRACT

The Ras superfamily comprises many guanine nucleotide-binding proteins (G proteins) that are essential to intracellular signal transduction. The guanine nucleotide-dependent intrinsic flexibility patterns of five G proteins were investigated in atomic detail through Molecular Dynamics simulations of the GDP- and GTP-bound states (S(GDP) and S(GTP), respectively). For all the considered systems, the intrinsic flexibility of S(GDP) was higher than that of S(GTP), suggesting that Guanine Exchange Factor (GEF) recognition and nucleotide switch require higher amplitude motions than effector recognition or GTP hydrolysis. Functional mode, dynamic domain, and interaction energy correlation analyses highlighted significant differences in the dynamics of small G proteins and Gα proteins, especially in the inactive state. Indeed, S(GDP) of Gα(t), is characterized by a more extensive energy coupling between nucleotide binding site and distal regions involved in GEF recognition compared to small G proteins, which attenuates in the active state. Moreover, mechanically distinct domains implicated in nucleotide switch could be detected in the presence of GDP but not in the presence of GTP. Finally, in small G proteins, functional modes are more detectable in the inactive state than in the active one and involve changes in solvent exposure of two highly conserved amino acids in switches I and II involved in GEF recognition. The average solvent exposure of these amino acids correlates in turn with the rate of GDP release, suggesting for them either direct or indirect roles in the process of nucleotide switch. Collectively, nucleotide binding changes the information flow through the conserved Ras-like domain, where GDP enhances the flexibility of mechanically distinct portions involved in nucleotide switch, and favors long distance allosteric communication (in Gα proteins), compared to GTP.


Subject(s)
Guanosine Diphosphate/metabolism , Guanosine Triphosphate/metabolism , Molecular Dynamics Simulation , ras Proteins/chemistry , ras Proteins/metabolism , Amino Acid Sequence , Binding Sites , Computational Biology , Guanosine Diphosphate/chemistry , Guanosine Triphosphate/chemistry , Molecular Sequence Data , Pliability , Principal Component Analysis , Protein Structure, Tertiary , Sequence Alignment
18.
Nucleic Acids Res ; 38(7): 2498-511, 2010 Apr.
Article in English | MEDLINE | ID: mdl-20071751

ABSTRACT

Hybrids of RNA with arabinonucleic acids 2'F-ANA and ANA have very similar structures but strikingly different thermal stabilities. We now present a thorough study combining NMR and other biophysical methods together with state-of-the-art theoretical calculations on a fully modified 10-mer hybrid duplex. Comparison between the solution structure of 2'F-ANA*RNA and ANA*RNA hybrids indicates that the increased binding affinity of 2'F-ANA is related to several subtle differences, most importantly a favorable pseudohydrogen bond (2'F-purine H8) which contrasts with unfavorable 2'-OH-nucleobase steric interactions in the case of ANA. While both 2'F-ANA and ANA strands maintained conformations in the southern/eastern sugar pucker range, the 2'F-ANA strand's structure was more compatible with the A-like structure of a hybrid duplex. No dramatic differences are found in terms of relative hydration for the two hybrids, but the ANA*RNA duplex showed lower uptake of counterions than its 2'F-ANA*RNA counterpart. Finally, while the two hybrid duplexes are of similar rigidities, 2'F-ANA single strands may be more suitably preorganized for duplex formation. Thus the dramatically increased stability of 2'F-ANA*RNA and ANA*RNA duplexes is caused by differences in at least four areas, of which structure and pseudohydrogen bonding are the most important.


Subject(s)
Arabinonucleotides/chemistry , RNA/chemistry , Thermodynamics , Cations/chemistry , Fluorine/chemistry , Hydrogen Bonding , Models, Molecular , Nuclear Magnetic Resonance, Biomolecular , Nucleic Acid Denaturation , Water/chemistry
19.
Nat Commun ; 13(1): 142, 2022 01 10.
Article in English | MEDLINE | ID: mdl-35013231

ABSTRACT

The establishment of cell identity during embryonic development involves the activation of specific gene expression programmes and is underpinned by epigenetic factors including DNA methylation and histone post-translational modifications. G-quadruplexes are four-stranded DNA secondary structures (G4s) that have been implicated in transcriptional regulation and cancer. Here, we show that G4s are key genomic structural features linked to cellular differentiation. We find that G4s are highly abundant in human embryonic stem cells and are lost during lineage specification. G4s are prevalent in enhancers and promoters. G4s that are found in common between embryonic and downstream lineages are tightly linked to transcriptional stabilisation of genes involved in essential cellular functions as well as transitions in the histone post-translational modification landscape. Furthermore, the application of small molecules that stabilise G4s causes a delay in stem cell differentiation, keeping cells in a more pluripotent-like state. Collectively, our data highlight G4s as important epigenetic features that are coupled to stem cell pluripotency and differentiation.


Subject(s)
Cell Lineage/genetics , Epigenesis, Genetic , G-Quadruplexes , Histones/metabolism , Human Embryonic Stem Cells/metabolism , Pluripotent Stem Cells/metabolism , Protein Processing, Post-Translational , Biomarkers/metabolism , Cell Differentiation , Cell Line , DNA/genetics , DNA/metabolism , DNA Methylation , Enhancer Elements, Genetic , Gene Expression , Histones/genetics , Human Embryonic Stem Cells/cytology , Humans , Nanog Homeobox Protein/genetics , Nanog Homeobox Protein/metabolism , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/metabolism , Nestin/genetics , Nestin/metabolism , Octamer Transcription Factor-3/genetics , Octamer Transcription Factor-3/metabolism , PAX6 Transcription Factor/genetics , PAX6 Transcription Factor/metabolism , Pluripotent Stem Cells/cytology , Promoter Regions, Genetic , Receptors, Nerve Growth Factor/genetics , Receptors, Nerve Growth Factor/metabolism , Transcription Factor AP-2/genetics , Transcription Factor AP-2/metabolism
20.
Nucleic Acids Res ; 37(17): 5589-601, 2009 Sep.
Article in English | MEDLINE | ID: mdl-19620215

ABSTRACT

Modified thrombin-binding aptamers carrying 2'-deoxyguanine (dG) residues with locked North- or South-bicyclo[3.1.0]hexane pseudosugars were synthesized. Individual 2'-deoxyguanosines at positions dG5, dG10, dG14 and dG15 of the aptamer were replaced by these analogues where the North/anti and South/syn conformational states were confined. It was found that the global structure of the DNA aptamer was, for the most part, very accommodating. The substitution at positions 5, 10 and 14 with a locked South/syn-dG nucleoside produced aptamers with the same stability and global structure as the innate, unmodified one. Replacing position 15 with the same South/syn-dG nucleoside induced a strong destabilization of the aptamer, while the antipodal North/anti-dG nucleoside was less destabilizing. Remarkably, the insertion of a North/anti-dG nucleoside at position 14, where both pseudosugar conformation and glycosyl torsion angle are opposite with respect to the native structure, led to the complete disruption of the G-tetraplex structure as detected by NMR and confirmed by extensive molecular dynamics simulations. We conclude that conformationally locked bicyclo[3.1.0]hexane nucleosides appear to be excellent tools for studying the role of key conformational parameters that are critical for the formation of a stable, antiparallel G-tetrad DNA structures.


Subject(s)
Aptamers, Nucleotide/chemistry , Deoxyguanosine/analogs & derivatives , Aptamers, Nucleotide/chemical synthesis , Carbohydrate Conformation , Circular Dichroism , Deoxyguanosine/chemistry , G-Quadruplexes , Models, Molecular , Nuclear Magnetic Resonance, Biomolecular , Nucleic Acid Conformation , Nucleic Acid Denaturation , Nucleic Acid Probes/chemistry , Nucleosides/chemical synthesis , Oligonucleotides/chemical synthesis , Temperature
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