C‐rich DNA has the capacity to form a tetra‐stranded structure known as an i‐motif. The i‐motifs within genomic DNA have been proposed to contribute to the regulation of DNA transcription. However, ...direct experimental evidence for the existence of these structures in vivo has been missing. Whether i‐motif structures form in complex environment of living cells is not currently known. Herein, using state‐of‐the‐art in‐cell NMR spectroscopy, we evaluate the stabilities of i‐motif structures in the complex cellular environment. We show that i‐motifs formed from naturally occurring C‐rich sequences in the human genome are stable and persist in the nuclei of living human cells. Our data show that i‐motif stabilities in vivo are generally distinct from those in vitro. Our results are the first to interlink the stability of DNA i‐motifs in vitro with their stability in vivo and provide essential information for the design and development of i‐motif‐based DNA biosensors for intracellular applications.
DNA i‐motif live coverage: The stabilities of DNA i‐motif structures formed from naturally occurring C‐rich sequences in the human genome were evaluated in the nuclei of living human cells. The reversibility of the i‐motif folding/unfolding cycle in vivo was demonstrated and the (thermodynamic) stability of DNA i‐motifs in vitro was interlinked with their stability in vivo.
DNA quadruplex structures provide an additional layer of regulatory control in genome maintenance and gene expression and are widely used in nanotechnology. We report the discovery of an ...unprecedented tetrastranded structure formed from a native G‐rich DNA sequence originating from the telomeric region of Caenorhabditis elegans. The structure is defined by multiple properties that distinguish it from all other known DNA quadruplexes. Most notably, the formation of a stable so‐called KNa‐quadruplex (KNaQ) requires concurrent coordination of K+ and Na+ ions at two distinct binding sites. This structure provides novel insight into G‐rich DNA folding under ionic conditions relevant to eukaryotic cell physiology and the structural evolution of telomeric DNA. It highlights the differences between the structural organization of human and nematode telomeric DNA, which should be considered when using C. elegans as a model in telomere biology, particularly in drug screening applications. Additionally, the absence/presence of KNaQ motifs in the host/parasite introduces an intriguing possibility of exploiting the KNaQ fold as a plausible antiparasitic drug target. The structure's unique shape and ion dependency and the possibility of controlling its folding by using low‐molecular‐weight ligands can be used for the design or discovery of novel recognition DNA elements and sensors.
A tetrastranded DNA structure, KNa‐quadruplex (KNaQ), is described. KNaQ forms from repetitive DNA sequences that are abundant in (parasitic) worms but extremely rare in humans or livestock. This opens a possibility of exploiting the fold as a plausible antiparasitic drug target. The structure's unique properties distinguish it from all other known DNA quadruplexes and can be used to design novel recognition DNA elements/sensors.
Genome-wide studies of DNA replication origins revealed that origins preferentially associate with an Origin G-rich Repeated Element (OGRE), potentially forming G-quadruplexes (G4). Here, we ...functionally address their requirements for DNA replication initiation in a series of independent approaches. Deletion of the OGRE/G4 sequence strongly decreased the corresponding origin activity. Conversely, the insertion of an OGRE/G4 element created a new replication origin. This element also promoted replication of episomal EBV vectors lacking the viral origin, but not if the OGRE/G4 sequence was deleted. A potent G4 ligand, PhenDC3, stabilized G4s but did not alter the global origin activity. However, a set of new, G4-associated origins was created, whereas suppressed origins were largely G4-free. In vitro Xenopus laevis replication systems showed that OGRE/G4 sequences are involved in the activation of DNA replication, but not in the pre-replication complex formation. Altogether, these results converge to the functional importance of OGRE/G4 elements in DNA replication initiation.
Recent studies indicate that i‐DNA, a four‐stranded cytosine‐rich DNA also known as the i‐motif, is actually formed in vivo; however, a systematic study on sequence effects on stability has been ...missing. Herein, an unprecedented number of different sequences (271) bearing four runs of 3–6 cytosines with different spacer lengths has been tested. While i‐DNA stability is nearly independent on total spacer length, the central spacer plays a special role on stability. Stability also depends on the length of the C‐tracts at both acidic and neutral pHs. This study provides a global picture on i‐DNA stability thanks to the large size of the introduced data set; it reveals unexpected features and allows to conclude that determinants of i‐DNA stability do not mirror those of G‐quadruplexes. Our results illustrate the structural roles of loops and C‐tracts on i‐DNA stability, confirm its formation in cells, and allow establishing rules to predict its stability.
i‐DNA is an emerging non‐canonical DNA secondary structure as an anticancer target and as a basic element in the programmable bionanotechnology. A large number of i‐DNA‐prone sequences were tested to disclose how primary sequences determine the i‐DNA stability both in vitro and in cells.
We compared here 80 different sequences containing four tracts of three guanines with loops of variable length (between 1 and 15 bases for unmodified sequences, up to 30 for fluorescently labeled ...oligonucleotides). All sequences were capable of forming stable quadruplexes, with Tm above physiological temperature in most cases. Unsurprisingly, the melting temperature was systematically lower in sodium than in potassium but the difference between both ionic conditions varied between 1 and >39°C (average difference: 18.3°C). Depending on the sequence context, and especially for G4 sequences involving two very short loops, the third one may be very long without compromising the stability of the quadruplex. A strong inverse correlation between total loop length and Tm was found in K⁺: each added base leads to a 2°C drop in Tm or ~0.3 kcal/mol loss in ΔG°. The trend was less clear in Na⁺, with a longer than expected optimal loop length (up to 5 nt). This study will therefore extend the sequence repertoire of quadruplex-prone sequences, arguing for a modification of the widely used consensus (maximal loop size of 7 bases).
The i‐motif DNA, also known as i‐DNA, is a non‐canonical DNA secondary structure formed by cytosine‐rich sequences, consisting of two intercalated parallel‐stranded duplexes held together by ...hemi‐protonated cytosine–cytosine+ (C:C+) base pairs. The growing interest in the i‐DNA structure as a target in anticancer therapy increases the need for tools for a rapid and meaningful interpretation of the spectroscopic data of i‐DNA samples. Herein, we analyzed the circular dichroism (CD) and thermal difference UV‐absorbance spectra (TDS) of 255 DNA sequences by means of multivariate data analysis, aiming at unveiling peculiar spectral regions that could be used as diagnostic features during the analysis of i‐DNA‐forming sequences.
i‐DNA is an emerging non‐canonical DNA secondary structure that represents a suitable target in anticancer therapy. A multivariate data analysis unveiled peculiar TDS and CD spectral regions that could be used for the structural determination of i‐DNA‐forming sequences.
Abstract
Motivation
Expanding research highlights the importance of guanine quadruplex structures. Therefore, easy-accessible tools for quadruplex analyses in DNA and RNA molecules are important for ...the scientific community.
Results
We developed a web version of the G4Hunter application. This new web-based server is a platform-independent and user-friendly application for quadruplex analyses. It allows retrieval of gene/nucleotide sequence entries from NCBI databases and provides complete characterization of localization and quadruplex propensity of quadruplex-forming sequences. The G4Hunter web application includes an interactive graphical data representation with many useful options including visualization, sorting, data storage and export.
Availability and implementation
G4Hunter web application can be accessed at: http://bioinformatics.ibp.cz.
Supplementary information
Supplementary data are available at Bioinformatics online.
AS1411 is a G-rich DNA oligonucleotide that functions as an aptamer of the protein nucleolin, found at high levels on the surface of cancer cells but not on the surface of normal cells. Herein, we ...have studied AS1411 as a supramolecular carrier for the delivery of an acridine-based G-quadruplex ligand, C
, to HeLa cancer cells. Two AS1411 derivatives, LNA-AS1411 and U-AS1411, were also tested, in an attempt to compare AS1411 pharmacological properties. The results showed that AS1411-C
complexation was made with great binding strength and that it lowered the ligand's cytotoxicity towards non-malignant cells. This effect was suggested to be due to a decreased internalization of the complexed versus free C
as shown by flow cytometry. The AS1411 derivatives, despite forming a stable complex with C
, lacked the necessary tumour-selective behaviour. The binding of C
to AS1411 G-quadruplex structure did not negatively affect the recognition of nucleolin by the aptamer. The AS1411-C
repressed c-MYC expression at the transcriptional level, possibly due to C
ability to stabilize the c-MYC promoter G-quadruplexes. Overall, this study demonstrates the usefulness of AS1411 as a supramolecular carrier of the G-quadruplex binder C
and the potential of using its tumour-selective properties for the delivery of ligands for cancer therapy.
DNA is prone to structural polymorphism: beyond the iconic Watson-Crick double helix, nucleic acids can adopt a number of unusual motifs, at least in vitro. Scientists around the world gather every ...two years to discuss two of these oddities: G-quadruplexes and i-DNA. The seventh international meeting on G-quadruplex Nucleic Acids was held in Changchun, in Jilin province of the P.R. of China, approx. 1000 km North-east of Beijing. Nearly 320 participants gathered from Asia, Europe, North America and Oceania. More than 80 talks and as many posters summarized our current knowledge of these unusual DNA and RNA structures. During this meeting, the creation of the G4 society was announced, in order to coordinate efforts and share tools and knowledge in our field (https://www.g4-society.org).
•Meeting report for the seventh International Meeting on Quadruplex Nucleic Acids, Changchun, China, 6–9 Sept 2019.•Over 300 scientists gathered to share their newest results in the Quadruplex field.•Recurrent themes were: structure and folding, applications to biotechnologies, formation in cells, proteins & ligands.
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