Nejvíce citovaný článek - PubMed ID 29266664
Evaluation of the Stability of DNA i-Motifs in the Nuclei of Living Mammalian Cells
Nucleic acids, molecules essential for all life, can adopt many alternative structures besides the well-known right-handed double helix, some of which have been reported to exist and function in vivo. One of the most appropriate methods for structural studies of nucleic acids is circular dichroism spectroscopy, utilizing structure-induced chirality due to the asymmetric winding of absorbing nucleobases. Using electronic CD and absorption spectroscopies in combination with melting experiments, we analyzed a conformational equilibrium between DNA double helix and two alternative conformations of nucleic acids, cytosine i-motifs and guanine quadruplexes, as a function of the primary structure of model G/C-rich sequences, containing blocks of G and C runs in particular DNA strands. This paper is a part of special issue dedicated to 70th anniversary of the Biophysical Institute of the Czech Academy of Sciences, where circular dichroism spectroscopy of nucleic acids has been used successfully and impactfully for many years.
- Klíčová slova
- Circular dichroism spectroscopy, Conformation equilibrium, Cytosine i-motif, DNA, Guanine quadruplex,
- Publikační typ
- časopisecké články MeSH
The insulin-linked polymorphic region is a variable number of tandem repeats region of DNA in the promoter of the insulin gene that regulates transcription of insulin. This region is known to form the alternative DNA structures, i-motifs and G-quadruplexes. Individuals have different sequence variants of tandem repeats and although previous work investigated the effects of some variants on G-quadruplex formation, there is not a clear picture of the relationship between the sequence diversity, the DNA structures formed, and the functional effects on insulin gene expression. Here we show that different sequence variants of the insulin linked polymorphic region form different DNA structures in vitro. Additionally, reporter genes in cellulo indicate that insulin expression may change depending on which DNA structures form. We report the crystal structure and dynamics of an intramolecular i-motif, which reveal sequences within the loop regions forming additional stabilising interactions that are critical to formation of stable i-motif structures. The outcomes of this work reveal the detail in formation of stable i-motif DNA structures, with potential for rational based drug design for compounds to target i-motif DNA.
- MeSH
- DNA * chemie genetika MeSH
- G-kvadruplexy * MeSH
- inzulin * chemie genetika MeSH
- konformace nukleové kyseliny MeSH
- krystalografie rentgenová MeSH
- lidé MeSH
- molekulární modely MeSH
- nukleotidové motivy MeSH
- polymorfismus genetický MeSH
- promotorové oblasti (genetika) * MeSH
- reportérové geny MeSH
- sekvence nukleotidů MeSH
- tandemové repetitivní sekvence genetika MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- DNA * MeSH
- inzulin * MeSH
I-Motifs (iM) are non-canonical DNA structures potentially forming in the accessible, single-stranded, cytosine-rich genomic regions with regulatory roles. Chromatin, protein interactions, and intracellular properties seem to govern iM formation at sites with i-motif formation propensity (iMFPS) in human cells, yet their specific contributions remain unclear. Using in-cell NMR with oligonucleotide iMFPS models, we monitor iM-associated structural equilibria in asynchronous and cell cycle-synchronized HeLa cells at 37 °C. Our findings show that iMFPS displaying pHT < 7 under reference in vitro conditions occur predominantly in unfolded states in cells, while those with pHT > 7 appear as a mix of folded and unfolded states depending on the cell cycle phase. Comparing these results with previous data obtained using an iM-specific antibody (iMab) reveals that cell cycle-dependent iM formation has a dual origin, and iM formation concerns only a tiny fraction (possibly 1%) of genomic sites with iM formation propensity. We propose a comprehensive model aligning observations from iMab and in-cell NMR and enabling the identification of iMFPS capable of adopting iM structures under physiological conditions in living human cells. Our results suggest that many iMFPS may have biological roles linked to their unfolded states.
- MeSH
- azidy * MeSH
- benzazepiny * MeSH
- DNA MeSH
- HeLa buňky MeSH
- lidé MeSH
- magnetická rezonanční tomografie * MeSH
- protilátky MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- 7-iodo-8-hydroxy-3-methyl-1-(4-azidophenyl)-2,3,4,5-tetrahydro-1H-3-benzazepine MeSH Prohlížeč
- azidy * MeSH
- benzazepiny * MeSH
- DNA MeSH
- protilátky MeSH
Metal ions are essential components for the survival of living organisms. For most species, intracellular and extracellular ionic conditions differ significantly. As G-quadruplexes (G4s) are ion-dependent structures, changes in the [Na+]/[K+] ratio may affect the folding of genomic G4s. More than 11000 putative G4 sequences in the human genome (hg19) contain at least two runs of three continuous cytosines, and these mixed G/C-rich sequences may form a quadruplex or a competing hairpin structure based on G-C base pairing. In this study, we examine how the [Na+]/[K+] ratio influences the structures of G/C-rich sequences. The natural G4 structure with a 9-nt long central loop, CEBwt, was chosen as a model sequence, and the loop bases were gradually replaced by cytosines. The series of CEB mutations revealed that the presence of cytosines in G4 loops does not prevent G4 folding or decrease G4 stability but increases the probability of forming a competing structure, either a hairpin or an intermolecular duplex. Slow conversion to the quadruplex in vitro (in a potassium-rich buffer) and cells was demonstrated by NMR. 'Shape-shifting' sequences may respond to [Na+]/[K+] changes with delayed kinetics.
Introducing the flow through the bioreactor has revolutionized in-cell NMR spectroscopy by prolonging the measurement time available to acquire spectral information about biomacromolecules in metabolically active cells. Bioreactor technology relies on immobilizer matrices, which secure cells in the active volume of the NMR coil and enable uniform perfusion of the growth medium, supplying fresh nutrients to the cells while removing toxic byproducts of their metabolism. The main drawbacks of commonly used matrices include the inability to recover intact cells post-measurement for additional analyses and/or requirements for specific operating temperatures. Here, we report on the development and characterization of a set of thermosensitive and nontoxic triblock copolymers based on poly(D,L-lactide)-b-poly(ethylene glycol)-b-poly(D,L-lactide) (PLA-PEG-PLA). Here, we show for the first time that these copolymers are suitable as immobilizer matrices for the acquisition of in-cell NMR spectra of nucleic acids and proteins over a commonly used sample temperature range of 15-40 °C and, importantly, allow recovery of cells after completion of in-cell NMR spectra acquisition. We compared the performances of currently used matrices in terms of cell viability (dye exclusion assays), cellular metabolism (1D 31P NMR), and quality of in-cell NMR spectra of two model biomacromolecules (hybrid double-stranded/i-motif DNA and ubiquitin). Our results demonstrate the suitability and advantages of PLA-PEG-PLA copolymers for application in bioreactor-assisted in-cell NMR.
- Klíčová slova
- Bioreactor, Cell immobilization, In-cell NMR, PLA-PEG-PLA, Thermosensitive hydrogel,
- MeSH
- bioreaktory MeSH
- DNA MeSH
- magnetická rezonanční spektroskopie MeSH
- nukleární magnetická rezonance biomolekulární MeSH
- nukleové kyseliny * MeSH
- polymery chemie MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- DNA MeSH
- nukleové kyseliny * MeSH
- polylactide-polyethylene glycol-polylactide MeSH Prohlížeč
- polymery MeSH
Cytosine-rich DNA regions can form four-stranded structures based on hemi-protonated C.C+ pairs, called i-motifs (iMs). Using CD, UV absorption, NMR spectroscopy, and DSC calorimetry, we show that model (CnT3)3Cn (Cn) sequences adopt iM under neutral or slightly alkaline conditions for n > 3. However, the iMs are formed with long-lasting kinetics under these conditions and melt with significant hysteresis. Sequences with n > 6 melt in two or more separate steps, indicating the presence of different iM species, the proportion of which is dependent on temperature and incubation time. At ambient temperature, kinetically favored iMs of low stability are formed, most likely consisting of short C.C+ blocks. These species act as kinetic traps and prevent the assembly of thermodynamically favored, fully C.C+ paired iMs. A higher temperature is necessary to unfold the kinetic forms and enable their substitution by a slowly developing thermodynamic structure. This complicated kinetic partitioning process considerably slows down iM folding, making it much slower than the timeframes of biological reactions and, therefore, unlikely to have any biological relevance. Our data suggest kinetically driven iM species as more likely to be biologically relevant than thermodynamically most stable iM forms.
- MeSH
- DNA * genetika chemie MeSH
- kinetika MeSH
- koncentrace vodíkových iontů MeSH
- konformace nukleové kyseliny MeSH
- nukleotidové motivy MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- DNA * MeSH
Berberine, the most known quaternary protoberberine alkaloid (QPA), has been reported to inhibit the SIK3 protein connected with breast cancer. Berberine also appears to reduce the bcl-2 and XIAP expression-proteins responsible for the inhibition of apoptosis. As some problems in the therapy with berberine arose, we studied the DNA binding properties of escholidine, another QPA alkaloid. CD, fluorescence, and NMR examined models of i-motif and G-quadruplex sequences present in the n-myc gene and the c-kit gene. We provide evidence that escholidine does not induce stabilization of the i-motif sequences, while the interaction with G-quadruplex structures appears to be more significant.
- Klíčová slova
- DNA, G-quadruplex, alkaloid, cancer, escholidine, spectroscopy,
- Publikační typ
- časopisecké články MeSH
Non-canonical forms of nucleic acids represent challenging objects for both structure-determination and investigation of their potential role in living systems. In this work, we uncover a structure adopted by GA repetition locked in a parallel homoduplex by an i-motif. A series of DNA oligonucleotides comprising GAGA segment and C3 clip is analyzed by NMR and CD spectroscopies to understand the sequence-structure-stability relationships. We demonstrate how the relative position of the homopurine GAGA segment and the C3 clip as well as single-base mutations (guanine deamination and cytosine methylation) affect base pairing arrangement of purines, i-motif topology and overall stability. We focus on oligonucleotides C3GAGA and methylated GAGAC3 exhibiting the highest stability and structural uniformity which allowed determination of high-resolution structures further analyzed by unbiased molecular dynamics simulation. We describe sequence-specific supramolecular interactions on the junction between homoduplex and i-motif blocks that contribute to the overall stability of the structures. The results show that the distinct structural motifs can not only coexist in the tight neighborhood within the same molecule but even mutually support their formation. Our findings are expected to have general validity and could serve as guides in future structure and stability investigations of nucleic acids.
Recently, the 1H-detected in-cell NMR spectroscopy has emerged as a unique tool allowing the characterization of interactions between nucleic acid-based targets and drug-like molecules in living human cells. Here, we assess the application potential of 1H and 19F-detected in-cell NMR spectroscopy to profile drugs/ligands targeting DNA G-quadruplexes, arguably the most studied class of anti-cancer drugs targeting nucleic acids. We show that the extension of the original in-cell NMR approach is not straightforward. The severe signal broadening and overlap of 1H in-cell NMR spectra of polymorphic G-quadruplexes and their complexes complicate their quantitative interpretation. Nevertheless, the 1H in-cell NMR can be used to identify drugs that, despite strong interaction in vitro, lose their ability to bind G-quadruplexes in the native environment. The in-cell NMR approach is adjusted to a recently developed 3,5-bis(trifluoromethyl)phenyl probe to monitor the intracellular interaction with ligands using 19F-detected in-cell NMR. The probe allows dissecting polymorphic mixture in terms of number and relative populations of individual G-quadruplex species, including ligand-bound and unbound forms in vitro and in cellulo. Despite the probe's discussed limitations, the 19F-detected in-cell NMR appears to be a promising strategy to profile G-quadruplex-ligand interactions in the complex environment of living cells.
- Klíčová slova
- BRACO19, Bcl2, G-quadruplex, KRAS, NMM, PhenDC3, drug, in-cell NMR, ligand, telomeric DNA,
- MeSH
- DNA chemie účinky léků MeSH
- G-kvadruplexy účinky léků MeSH
- konformace nukleové kyseliny účinky léků MeSH
- léčivé přípravky chemie MeSH
- lidé MeSH
- ligandy MeSH
- magnetická rezonanční spektroskopie MeSH
- molekulární modely MeSH
- protony MeSH
- vazebná místa účinky léků MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- DNA MeSH
- léčivé přípravky MeSH
- ligandy MeSH
- protony MeSH
We recently showed that Saccharomyces cerevisiae telomeric DNA can fold into an unprecedented pseudocircular G-hairpin (PGH) structure. However, the formation of PGHs in the context of extended sequences, which is a prerequisite for their function in vivo and their applications in biotechnology, has not been elucidated. Here, we show that despite its 'circular' nature, PGHs tolerate single-stranded (ss) protrusions. High-resolution NMR structure of a novel member of PGH family reveals the atomistic details on a junction between ssDNA and PGH unit. Identification of new sequences capable of folding into one of the two forms of PGH helped in defining minimal sequence requirements for their formation. Our time-resolved NMR data indicate a possibility that PGHs fold via a complex kinetic partitioning mechanism and suggests the existence of K+ ion-dependent PGH folding intermediates. The data not only provide an explanation of cation-type-dependent formation of PGHs, but also explain the unusually large hysteresis between PGH melting and annealing noted in our previous study. Our findings have important implications for DNA biology and nanotechnology. Overrepresentation of sequences able to form PGHs in the evolutionary-conserved regions of the human genome implies their functionally important biological role(s).
- MeSH
- konformace nukleové kyseliny MeSH
- kruhová DNA chemie MeSH
- molekulární modely MeSH
- nukleární magnetická rezonance biomolekulární MeSH
- nukleotidové motivy MeSH
- párování bází MeSH
- Saccharomyces cerevisiae genetika MeSH
- stereoizomerie MeSH
- telomery chemie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- kruhová DNA MeSH
