Noncoding RNAs are functional transcripts that are not translated into proteins. They represent the largest portion of the human transcriptome and have been shown to regulate gene expression networks in both physiological and pathological cell conditions. Research in this field has made remarkable progress in the comprehension of how aberrations in noncoding RNA drive relevant disease-associated phenotypes; however, the biological role and mechanism of action of several noncoding RNAs still need full understanding. Besides fulfilling its function through sequence-based mechanisms, RNA can form complex secondary and tertiary structures which allow non-canonical interactions with proteins and/or other nucleic acids. In this context, the presence of G-quadruplexes in microRNAs and long noncoding RNAs is increasingly being reported. This evidence suggests a role for RNA G-quadruplexes in controlling microRNA biogenesis and mediating noncoding RNA interaction with biological partners, thus ultimately regulating gene expression. Here, we review the state of the art of G-quadruplexes in the noncoding transcriptome, with their structural and functional characterization. In light of the existence and further possible development of G-quadruplex binders that modulate G-quadruplex conformation and protein interactions, we also discuss the therapeutic potential of G-quadruplexes as targets to interfere with disease-associated noncoding RNAs.

G-quadruplexes are four-stranded conformations of nucleic acids that act as cellular epigenetic regulators. A dynamic G-quadruplex forming region in the HIV-1 LTR promoter represses HIV-1 transcription when in the folded conformation. This activity is enhanced by nucleolin, which induces and stabilizes the HIV-1 LTR G-quadruplexes. In this work by a combined pull-down/mass spectrometry approach, we consistently found hnRNP A2/B1 as an additional LTR-G-quadruplex interacting protein. Surface plasmon resonance confirmed G-quadruplex specificity over linear sequences and fluorescence resonance energy transfer analysis indicated that hnRNP A2/B1 is able to efficiently unfold the LTR G-quadruplexes. Evaluation of the thermal stability of the LTR G-quadruplexes in different-length oligonucleotides showed that the protein is fit to be most active in the LTR full-length environment. When hnRNP A2/B1 was silenced in cells, LTR activity decreased, indicating that the protein acts as a HIV-1 transcription activator. Our data highlight a tightly regulated control of transcription based on G-quadruplex folding/unfolding, which depends on interacting cellular proteins. These findings provide a deeper understanding of the viral transcription mechanism and may pave the way to the development of drugs effective against the integrated HIV-1, present both in actively and latently infected cells.

Targeting of G-quadruplexes, non-canonical conformations that form in G-rich regions of nucleic acids, has been proposed as a novel therapeutic strategy toward several diseases, including cancer and infections. The unavailability of highly selective molecules targeting a G-quadruplex of choice has hampered relevant applications. Herein, we describe a novel approach, based on naphthalene diimide (NDI)-peptide nucleic acid (PNA) conjugates, taking advantage of the cooperative interaction of the NDI with the G-quadruplex structure and hybridization of the PNA with the flanking region upstream or downstream the targeted G-quadruplex. By biophysical and biomolecular assays, we show that the NDI-PNA conjugates are able to specifically recognize the G-quadruplex of choice within the HIV-1 LTR region, consisting of overlapping and therefore mutually exclusive G-quadruplexes. Additionally, the conjugates can induce and stabilize the least populated G-quadruplex at the expenses of the more stable ones. The general and straightforward design and synthesis, which readily apply to any G4 target of choice, together with both the red-fluorescent emission and the possibility to introduce cellular localization signals, make the novel conjugates available to selectively control G-quadruplex folding over a wide range of applications.

Well-differentiated liposarcoma (WDLPS) is a malignant neoplasia hard to diagnose and treat. Its main molecular signature is amplification of the MDM2-containing genomic region. The MDM2 oncogene is the master regulator of p53: its overexpression enhances p53 degradation and inhibits apoptosis, leading to the tumoral phenotype. Here, we show that the MDM2 inducible promoter G-rich region folds into stable G-quadruplexes both in vitro and in vivo and it is specifically recognized by cellular helicases. Cell treatment with G-quadruplex-ligands reduces MDM2 expression and p53 degradation, thus stimulating cancer cell cycle arrest and apoptosis. Structural characterization of the MDM2 G-quadruplex revealed an extraordinarily stable, unique four-tetrad antiparallel dynamic conformation, amenable to selective targeting. These data indicate the feasibility of an out-of-the-box G-quadruplex-targeting approach to defeat WDLPS and all tumours where restoration of wild-type p53 is sought. They also point to G-quadruplex-dependent genomic instability as possible cause of MDM2 expansion and WDLPS tumorigenesis.

Retroviruses infect almost all vertebrates, from humans to domestic and farm animals, from primates to wild animals, where they cause severe diseases, including immunodeficiencies, neurological disorders and cancer. Non-human retroviruses have also been recently associated to human diseases. To date no effective treatments are available, therefore finding retrovirus-specific therapeutic targets is becoming an impellent issue. G-quadruplexes are four-stranded nucleic acid structures that form in guanine-rich regions. Highly conserved G-quadruplexes located in the long terminal repeat (LTR) promoter of HIV-1 were shown to modulate the virus transcription machinery: moreover, the astonishingly high degree of conservation of G-quadruplex sequences in all primate lentiviruses corroborates the idea that these non-canonical nucleic acid structures are crucial elements in the lentiviral biology and thus have been selected for during evolution. In this work we aimed at investigating the presence and conservation of G-quadruplexes in the Retroviridae family. Genome-wide bioinformatics analysis showed that, despite their documented high genetic variability, most retroviruses contain highly conserved putative G-quadruplex forming sequences in their promoter region. Biophysical and biomolecular assays proved that these sequences actually fold into G-quadruplex in physiological concentration of relevant cations and that they are further stabilized by ligands. These results validate the relevance of G-quadruplexes in retroviruses and endorse the employment of G-quadruplex ligands as innovative antiretroviral drugs. This study indicates new possible pathways in the management of retroviral infections in humans and animal species. Moreover, it may shed light on the mechanism and functions of retrovirus genomes and derived transposable elements in the human genome.