DNA has played an early and powerful role in the development of bottom-up nanotechnologies, not least because of DNA’s precise, predictable, and controllable properties of assembly on the nanometer ...scale. Watson–Crick complementarity has been used to build complex 2D and 3D architectures and design a number of nanometer-scale systems for molecular computing, transport, motors, and biosensing applications. Most of such devices are built with classical B-DNA helices and involve classical A-T/U and G-C base pairs. However, in addition to the above components underlying the iconic double helix, a number of alternative pairing schemes of nucleobases are known. This review focuses on two of these noncanonical classes of DNA helices: G-quadruplexes and the i-motif. The unique properties of these two classes of DNA helix have been utilized toward some remarkable constructions and applications: G-wires; nanostructures such as DNA origami; reconfigurable structures and nanodevices; the formation and utilization of hemin-utilizing DNAzymes, capable of generating varied outputs from biosensing nanostructures; composite nanostructures made up of DNA as well as inorganic materials; and the construction of nanocarriers that show promise for the therapeutics of diseases.
Critical evidence for the biological relevance of G-quadruplexes (G4) has recently been obtained in seminal studies performed in a variety of organisms. Four-stranded G-quadruplex DNA structures are ...promising drug targets as these non-canonical structures appear to be involved in a number of key biological processes. Given the growing interest for G4, accurate tools to predict G-quadruplex propensity of a given DNA or RNA sequence are needed. Several algorithms such as Quadparser predict quadruplex forming propensity. However, a number of studies have established that sequences that are not detected by these tools do form G4 structures (false negatives) and that other sequences predicted to form G4 structures do not (false positives). Here we report development and testing of a radically different algorithm, G4Hunter that takes into account G-richness and G-skewness of a given sequence and gives a quadruplex propensity score as output. To validate this model, we tested it on a large dataset of 392 published sequences and experimentally evaluated quadruplex forming potential of 209 sequences using a combination of biophysical methods to assess quadruplex formation in vitro. We experimentally validated the G4Hunter algorithm on a short complete genome, that of the human mitochondria (16.6 kb), because of its relatively high GC content and GC skewness as well as the biological relevance of these quadruplexes near instability hotspots. We then applied the algorithm to genomes of a number of species, including humans, allowing us to conclude that the number of sequences capable of forming stable quadruplexes (at least in vitro) in the human genome is significantly higher, by a factor of 2-10, than previously thought.
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.
G‐quadruplexes (G4) play crucial roles in biology, analytical chemistry and nanotechnology. The stability of G4 structures is impacted by the number of G‐quartets, the length and positions of loops, ...flanking motifs, as well as additional structural elements such as bulges, capping base pairs, or triads. Algorithms such as G4Hunter or Quadparser may predict if a given sequence is G4‐prone by calculating a quadruplex propensity score; however, experimental validation is still required. We previously demonstrated that this validation is not always straightforward, and that a combination of techniques is often required to unambiguously establish whether a sequence forms a G‐quadruplex or not. In this article, we adapted the well‐known FRET‐melting assay to characterize G4 in batch, where the sequence to be tested is added, as an unlabeled competitor, to a system composed of a dual‐labeled probe (F21T) and a specific quadruplex ligand. PhenDC3 was preferred over TMPyP4 because of its better selectivity for G‐quadruplexes. In this so‐called FRET‐MC (melting competition) assay, G4‐forming competitors lead to a marked decrease of the ligand‐induced stabilization effect (∆Tm), while non‐specific competitors (e.g., single‐ or double‐stranded sequences) have little effect. Sixty‐five known sequences with different typical secondary structures were used to validate the assay, which was subsequently employed to assess eight novel sequences that were not previously characterized.
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.
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.
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.
Guanine-rich sequences can form G-quadruplexes (G4) in living cells, making these structures promising anti-cancer targets. Compounds able to recognize these structures have been investigated as ...potential anticancer drugs; however, no G4 binder has yet been approved in the clinic. Here, we describe G4 ligands structure-activity relationships, in vivo effects as well as clinical trials. Addressing G4 ligand characteristics, targeting challenges, and structure-activity relationships, this review provides insights into the development of potent and selective G4-targeting molecules for therapeutic applications.
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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.
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