Triplex‐Forming Twisted Intercalating Nucleic Acids (TINAs): Design Rules,Stabilization of Antiparallel DNA Triplexes and Inhibition of G‐Quartet‐Dependent Self‐Association

The majority of studies on DNA triple helices have been focused on pH‐sensitive parallel triplexes with Hoogsteen CT‐containing third strands that require protonation of cytosines. Reverse Hoogsteen GT/GA‐containing antiparallel triplex‐forming oligonucleotides (TFOs) do not require an acidic pH but their applicability in triplex technology is limited because of their tendency to form undesired highly stable aggregates such as G‐quadruplexes. In this study, G‐rich oligonucleotides containing 2–4 insertions of twisted intercalating nucleic acid (TINA) monomers are demonstrated to disrupt the formation of G‐quadruplexes and form stable antiparallel triplexes with target DNA duplexes. The structure of TINA‐incorporated oligonucleotides was optimized, the rules of their design were established and the optimal triplex‐forming oligonucleotides were selected. These oligonucleotides show high affinity towards a 16 bp homopurine model sequence from the HIV‐1 genome; dissociation constants as low as 160 nM are observed whereas the unmodified TFO does not show any triplex formation and instead forms an intermolecular G‐quadruplex with T_m exceeding 90 °C in the presence of 50 mM NaCl. Here we present a set of rules that help to reach the full potential of TINA‐TFOs and demonstrate the effect of TINA on the formation and stability of triple helical DNA.     

DNA and RNA Quadruplex-Binding Proteins

Four-stranded DNA structures were structurally characterized in vitro by NMR, X-ray and Circular Dichroism spectroscopy in detail. Among the different types of quadruplexes (i-Motifs, minor groove quadruplexes, G-quadruplexes, etc.), the best described are G-quadruplexes which are featured by Hoogsteen base-paring. Sequences with the potential to form quadruplexes are widely present in genome of all organisms. They are found often in repetitive sequences such as telomeric ones, and also in promoter regions and 5'' non-coding sequences. Recently, many proteins with binding affinity to G-quadruplexes have been identified. One of the initially portrayed G-rich regions, the human telomeric sequence (TTAGGG)_n, is recognized by many proteins which can modulate telomerase activity. Sequences with the potential to form G-quadruplexes are often located in promoter regions of various oncogenes. The NHE III₁ region of the c-MYC promoter has been shown to interact with nucleolin protein as well as other G-quadruplex-binding proteins. A number of G-rich sequences are also present in promoter region of estrogen receptor alpha. In addition to DNA quadruplexes, RNA quadruplexes, which are critical in translational regulation, have also been predicted and observed. For example, the RNA quadruplex formation in telomere-repeat-containing RNA is involved in interaction with TRF2 (telomere repeat binding factor 2) and plays key role in telomere regulation. All these fundamental examples suggest the importance of quadruplex structures in cell processes and their understanding may provide better insight into aging and disease development.     

Fluorine‐Mediated Editing of a G‐Quadruplex Folding Pathway

A (3+1)‐hybrid‐type G‐quadruplex was substituted within its central tetrad by a single 2′‐fluoro‐modified guanosine. Driven by the anti‐favoring nucleoside analogue, a novel quadruplex fold with inversion of a single G‐tract and conversion of a propeller loop into a lateral loop emerges. In addition, scalar couplings across hydrogen bonds demonstrate the formation of intra‐ and inter‐residual F ??? H8?C8 pseudo‐hydrogen bonds within the modified quadruplexes. Alternative folding can be rationalized by the impact of fluorine on intermediate species on the basis of a kinetic partitioning mechanism. Apparently, chemical or other environmental perturbations are able to redirect folding of a quadruplex, possibly modulating its regulatory role in physiological processes.     

Inhibition of dicing of guanosine-rich shRNAs by quadruplex-binding compounds

RNA interference is triggered by small hairpin precursors that are processed by the endonuclease dicer to yield active species such as siRNAs and miRNAs. To regulate the RNAi-mediated suppression of gene expression, we imagined a strategy that relies on the sequence-specific inhibition of shRNA precursor processing by immediate RNA-small molecule interactions. Here, we present a first step in this direction by augmenting shRNAs with guanosine-rich sequences that are prone to fold into four-stranded structures. The addition of small molecules that selectively bind to such quadruplex sequences should allow for the specific inhibition of dicing of shRNAs that contain suitable G-rich elements. In an attempt to find compounds that protect against dicer processing, we have examined the effects of quadruplex-binding compounds on the dicer processing of shRNAs containing G-quadruplexes. Although a variety of small molecules that are known to bind to quadruplexes inhibited in vitro dicing of shRNAs, only two substance classes, namely certain porphyrazines and bisquinolinium compounds, showed selective inhibition of G-rich shRNAs compared to control sequences lacking guanine-rich elements. The G-rich shRNAs displayed a potent knockdown of gene expression in mammalian cell culture, but the effect was not influenced by addition of the respective quadruplex-binding compounds.     

Fluorescent Probes for G‐Quadruplex Structures

Mounting evidence supports the presence of biologically relevant G‐quadruplexes in single‐cell organisms, but the existence of endogenous G‐quadruplex structures in mammalian cells remains highly controversial. This is due, in part, to the common misconception that DNA and RNA molecules are passive information carriers with relatively little structural or functional complexity. For those working in the field, however, the lack of available tools for characterizing DNA structures in vivo remains a major limitation to addressing fundamental questions about structure–function relationships of nucleic acids. In this review, we present progress towards the direct detection of G‐quadruplex structures by using small molecules and modified oligonucleotides as fluorescent probes. While most development has focused on cell‐permeable probes that selectively bind to G‐quadruplex structures with high affinity, these same probes can induce G‐quadruplex folding, thereby making the native conformation of the DNA or RNA molecule (i.e., in the absence of probe) uncertain. For this reason, modified oligonucleotides and fluorescent base analogues that serve as “internal” fluorescent probes are presented as an orthogonal means for detecting conformational changes, without necessarily perturbing the equilibria between G‐quadruplex, single‐stranded, and duplex DNA. The major challenges and motivation for the development of fluorescent probes for G‐quadruplex structures are presented, along with a summary of the key photophysical, biophysical, and biological properties of reported examples.     

Binding Properties of RNA Quadruplex of SARS-CoV-2 to Berberine Compared to Telomeric DNA Quadruplex

Previous studies suggest that berberine, an isoquinoline alkaloid, has antiviral potential and is a possible therapeutic candidate against SARS-CoV-2. The molecular underpinnings of its action are still unknown. Potential targets include quadruplexes (G4Q) in the viral genome as they play a key role in modulating the biological activity of viruses. While several DNA-G4Q structures and their binding properties have been elucidated, RNA-G4Qs such as RG-1 of the N-gene of SARS-CoV-2 are less explored. Using biophysical techniques, the berberine binding thermodynamics and the associated conformational and hydration changes of RG-1 could be characterized and compared with human telomeric DNA-G4Q 22AG. Berberine can interact with both quadruplexes. Substantial changes were observed in the interaction of berberine with 22AG and RG-1, which adopt different topologies that can also change upon ligand binding. The strength of interaction and the thermodynamic signatures were found to dependent not only on the initial conformation of the quadruplex, but also on the type of salt present in solution. Since berberine has shown promise as a G-quadruplex stabilizer that can modulate viral gene expression, this study may also contribute to the development of optimized ligands that can discriminate between binding to DNA and RNA G-quadruplexes.     

G‐Quadruplexes as Tools for Synthetic Biology

With the potential to engineer biological systems, synthetic biology is an emerging field that combines various disciplines of sciences. It encompasses combinations of DNA, RNA and protein modules for constructing desired systems and the “rewiring” of existing signalling networks. Despite recent advances, this field still lags behind in the artificial reconstruction of cellular processes, and thus demands new modules and switches to create “genetic circuits”. The widely characterised noncanonical nucleic acid secondary structures, G‐quadruplexes are promising candidates to be used as biological modules in synthetic biology. Structural plasticity and functional versatility are significant G‐quadruplex traits for its integration into a biological system and for diverse applications in synthetic circuits.     

Development of Fluorescent Protein Probes Specific for Parallel DNA and RNA G‐Quadruplexes

We have developed fluorescent protein probes specific for parallel G‐quadruplexes by attaching cyan fluorescent protein to the G‐quadruplex‐binding motif of the RNA helicase RHAU. Fluorescent probes containing RHAU peptide fragments of different lengths were constructed, and their binding to G‐quadruplexes was characterized. The selective recognition and discrimination of G‐quadruplex topologies by the fluorescent protein probes was easily detected by the naked eye or by conventional gel imaging.     

Flavin Conjugates for Delivery of Peptide Nucleic Acids

Oligonucleotides and their analogues, such as peptide nucleic acids (PNAs), can be used in chemical strategies to artificially control gene expression. Inefficient cellular uptake and inappropriate cellular localization still remain obstacles in biological applications, however, especially for PNAs. Here we demonstrate that conjugation of PNAs to flavin resulted in efficient internalization into cells through an endocytic pathway. The flavin–PNAs exhibited antisense activity in the sub‐micromolar range, in the context of a treatment facilitating endosomal escape. Increased endosomal release of flavin conjugates into the cytoplasm and/or nucleus was shown by chloroquine treatment and also—when the flavin–PNA was conjugated to rhodamine, a mild photosensitizer—upon light irradiation. In conclusion, an isoalloxazine moiety can be used as a carrier and attached to a cargo biomolecule, here a PNA, for internalization and functional cytoplasmic/nuclear delivery. Our findings could be useful for further design of PNAs and other oligonucleotide analogues as potent antisense agents.     

Bioinformatic Tools for the Analysis and Prediction of ncRNA Interactions

Noncoding RNAs (ncRNAs) play prominent roles in the regulation of gene expression via their interactions with other biological molecules such as proteins and nucleic acids. Although much of our knowledge about how these ncRNAs operate in different biological processes has been obtained from experimental findings, computational biology can also clearly substantially boost this knowledge by suggesting possible novel interactions of these ncRNAs with other molecules. Computational predictions are thus used as an alternative source of new insights through a process of mutual enrichment because the information obtained through experiments continuously feeds through into computational methods. The results of these predictions in turn shed light on possible interactions that are subsequently validated experimentally. This review describes the latest advances in databases, bioinformatic tools, and new in silico strategies that allow the establishment or prediction of biological interactions of ncRNAs, particularly miRNAs and lncRNAs. The ncRNA species described in this work have a special emphasis on those found in humans, but information on ncRNA of other species is also included.