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DNA sequence composition determines the topology and stability of G-quadruplexes (G4s). Bulged G-quadruplex structures (G4-Bs) are a subset of G4s characterized by 3D conformations with bulges. Current search algorithms fail to capture stable G4-B, making their genome-wide study infeasible. Here, we introduced a large family of computationally defined and experimentally verified potential G4-B forming sequences (pG4-BS). We found 478 263 pG4-BS regions that do not overlap 'canonical' G4-forming sequences in the human genome and are preferentially localized in transcription regulatory regions including R-loops and open chromatin. Over 90% of protein-coding genes contain pG4-BS in their promoter or gene body. We observed generally higher pG4-BS content in R-loops and their flanks, longer genes that are associated with brain tissue, immune and developmental processes. Also, the presence of pG4-BS on both template and non-template strands in promoters is associated with oncogenesis, cardiovascular disease and stemness. Our G4-BS models predicted G4-forming ability in vitro with 91.5% accuracy. Analysis of G4-seq and CUT&Tag data strongly supports the existence of G4-BS conformations genome-wide. We reconstructed a novel G4-B 3D structure located in the E2F8 promoter. This study defines a large family of G4-like sequences, offering new insights into the essential biological functions and potential future therapeutic uses of G4-B. Agency for Science, Technology and Research (A*STAR) Nanyang Technological University Published version Bioinformatics Institute, Biomedical Institutes/A-STAR, Singapore (in part); V.A.K. was supported by a SUNY EMPIRE innovation program scholar grant; Upstate Medical University Cancer Center grant; Upstate Foundation Turn4ACure Fund; research in A.T.P. lab was supported by Nanyang Technological University Singapore; P.J. was supported by the Office of the Permanent Secretary, Ministry of Higher Education, Science, Research and Innovation (OPS MHESI); Thailand Science Research and Innovation (TSRI) [RGNS 64-161]. Funding for open access charge: SUNY EMPIRE innovation program scholar grant, the Upstate Medical University Cancer Center grant; Upstate Foundation Turn4ACure Fund.

G-quadruplexes (G4s) are secondary structures abundant in DNA that may play regulatory roles in cells. Despite the ubiquity of the putative G-quadruplex sequences (PQS) in the human genome, only a small fraction forms secondary structures in cells. Folded G4, histone methylation and chromatin accessibility are all parts of the complexcisregulatory landscape. We propose an approach for G4 formation prediction in cells that incorporates epigenetic and chromatin accessibility data. The novel approach termedepiG4NNefficiently predicts cell-specific G4 formation in live cells based on a local epigenomic snapshot. Our architecture confirms the close relationship between H3K4me3 histone methylation, chromatin accessibility and G4 structure formation. Trained on A549 cell data,epiG4NNwas then able to predict G4x formation in HEK293T and K562 cell lines. We observe the dependency of model performance with different epigenetic features on the underlying experimental condition of G4 detection. We expect that this approach will contribute to the systematic understanding of correlations between structural and epigenomic feature landscape.

G-quadruplexes (G4s) are attractive anticancer targets. While right-handed G4s have been extensively investigated with many specific ligands reported, left-handed G4s formed by natural DNA have been recently discovered. Here we show that ligands specific for right-handed G4s, such as Phen-DC3 and RHAU peptide, do not bind specifically to left-handed G4s. In right-handed G4s, these ligands can displace capping overhangs and/or loops to stack on the exposed terminal tetrads. In contrast, the presence of tight T-capping in left-handed G4s hinders access to the tetrads. Ministry of Education (MOE) This work was supported by the Singapore Ministry of Education Academic Research Fund Tier 2 (MOE2015-T2-1- 092).

Acute Myeloid Leukaemia (AML) is the most common blood cancer in adults. Although 2 out of 3 AML patients go into total remission after chemotherapies and targeted therapies, the disease recurs in 60%-65% of younger adult patients within 3 years after diagnosis with a dramatically decreased survival rate. Therapeutic oligonucleotides are promising treatments under development for AML as they can be designed to silence oncogenes with high specificity and flexibility. However, there are not many well validated approaches for safely and efficiently delivering oligonucleotide drugs. This issue could be resolved by utilizing a new generation of delivery vehicles such as extracellular vesicles (EVs).In this study, we harness red blood cell-derived EVs (RBCEVs) and engineer them via exogenous drug loading and surface functionalization to develop an efficient drug delivery system for AML. Particularly, EVs are designed to target CD33, a common surface marker with elevated expression in AML cells via the conjugation of a CD33-binding monoclonal antibody onto the EV surface.The conjugation of RBCEVs with the CD33-binding antibody significantly increases the uptake of RBCEVs by CD33-positive AML cells, but not by CD33-negative cells. We also load CD33-targeting RBCEVs with antisense oligonucleotides (ASOs) targeting FLT3-ITD or miR-125b, 2 common oncogenes in AML, and demonstrate that the engineered EVs improve leukaemia suppression in in vitro and in vivo models of AML.Targeted RBCEVs represent an innovative, efficient, and versatile delivery platform for therapeutic ASOs and can expedite the clinical translation of oligonucleotide drugs for AML treatments by overcoming current obstacles in oligonucleotide delivery.

Aberrant expansion of the hexanucleotide GGGGCC (or G₄C₂) repeat in the human C9ORF72 gene is the most common genetic factor found behind amyotrophic lateral sclerosis and frontotemporal dementia. The hypothesized pathways, through which the repeat expansions contribute to the pathology, involve one or more secondary structural forms of the DNA and/or RNA sequences, such as G-quadruplexes, duplexes, and hairpins. Here, we study the structures of DNA and RNA duplexes formed by G₄C₂ repeats, which contain G(syn)·G(anti) base pairs flanked by either G·C or C·G base pairs. We show that duplexes formed by G₄C₂ repeats contain alternately two types of G·G pair contexts exhibiting different syn–anti base flipping dynamics (∼100 ms vs ∼2 ms for DNA and ∼50 ms vs ∼20 ms for RNA at 10 °C, respectively) depending on the flanking bases, with the slow-flipping G·G pairs being flanked by a guanine at the 5′-end and the fast-flipping G·G pairs being flanked by a cytosine at the 5′-end. Our findings on the structures and dynamics of G·G base pairs in DNA and RNA duplexes formed by G₄C₂ repeats provide a foundation for further studies of the functions and targeting of such biologically relevant motifs.

Dysregulated T-cell activation is a hallmark of several autoimmune diseases such as rheumatoid arthritis (RA) and multiple sclerosis (MS). The lymphocyte cytosolic protein 2 (LCP2), also known as SLP-76, is essential for the development and activation of T cells. Despite the critical role of LCP2 in T-cell activation and the need for developing drugs that modify T-cell activation, no LCP2 inhibitors have been developed. This can be explained by the "undruggable" nature of LCP2, lacking a structure permissive to standard small molecule inhibitor modalities. Here, we explored an alternative drug modality, developing antisense oligonucleotides (ASOs) targeting LCP2 mRNAs, and evaluated its activity in modulating T-cell activation. We identified a set of 3' UTR targeting LCP2 ASOs, which knocked down LCP2 in a human T-cell line and primary human T cells and found that these suppressed T-cell receptor mediated activation. We also found that the ASOs suppressed FcεR1-mediated mast cell activation, in line with the role of LCP2 in mast cells. Taken together, our data provide examples of how immunomodulatory ASOs that interfere with undruggable targets can be developed and propose that such drug modalities can be used to treat autoimmune diseases. Nanyang Technological University Published version This research was funded by grants from the Karolinska Institutet, Swedish Research Council, Swedish Cancer Society, Stiftelsen Professor Nanna Svartz Fond, the China Scholarship Council, and the Nanyang Technological University–Karolinska Institutet Joint PhD Programme. This work has additionally received support via the European Union/ European Federation of Pharmaceutical Industries and Associations (EU/EFPIA) Innovative Medicines Initiative Joint Undertaking (RTCure Grant 777357).

93del is a 16-nucleotide G-quadruplex-forming aptamer which can inhibit the activity of the HIV-1 integrase enzyme at nanomolar concentration. Previous structural analyses of 93del using NMR spectroscopy have shown that the aptamer forms an interlocked G-quadruplex structure in K+ solution. Due to its exceptional stability and unique topology, 93del has been used in many different studies involving DNA G-quadruplexes, such as DNA aptamer and multimer design, as well as DNA fluorescence research. To gain further insights on the structure of this unique aptamer, we have determined several high-resolution crystal structures of 93del and its variants. While confirming the overall dimeric interlocked G-quadruplex folding topology previously determined by NMR, our results reveal important detailed structural information, particularly the formation of a water-mediated A•G•G•G•G pentad. These insights allow us to better understand the formation of various structural elements in G-quadruplexes and should be useful for designing and manipulating G-quadruplex scaffolds with desired properties. Ministry of Education (MOE) National Research Foundation (NRF) Submitted/Accepted version This work was supported by Singapore National Research Foundation Investigatorship (NRF-NRFI2017-09) and Singapore Ministry of Education Academic Research Fund Tier 2 (MOE2018-T2-2-029).

G-quadruplex (G4) is a non-canonical four-stranded nucleic acid structure and the RHAU helicase has been identified to have high specificity for recognition of parallel-stranded G4s. We have designed and synthesized two stapled peptide analogues of the G4-specfic motif of RHAU, which preserve the G4 binding ability. Characterization of these peptides identified the stapled variants to exhibit higher helical formation propensity in aqueous buffer in comparison to the native RHAU sequence. Moreover, the stapled peptides exhibit superior enzymatic stability towards α-chymotrypsin. Our stapled RHAU peptides can serve as a new tool for targeting G4 nucleic acid structures. Ministry of Education (MOE) National Research Foundation (NRF) Accepted version This work was supported by the Singapore National Research Foundation Investigatorship (NRF-NRFI2017-09) and Singapore Ministry of Education Academic Research Fund Tier 2 (MOE2018-T2-2-029). No competing financial interests have been declared.

The triple-negative breast cancer (TNBC), a subtype of breast cancer which lacks of targeted therapies, exhibits a poor prognosis. It was shown recently that the PIM1 oncogene is highly related to the proliferation of TNBC cells. A quadruplex–duplex hybrid (QDH) forming sequence was recently found to exist near the transcription start site of PIM1. This structure could be an attractive target for regulation of the PIM1 gene expression and thus the treatment of TNBC. Here, we present the solution structures of two QDHs that could coexist in the human PIM1 gene. Form 1 is a three-G-tetrad-layered (3+1) G-quadruplex containing a propeller loop, a lateral loop and a stem-loop made up of three G•C Watson–Crick base pairs. On the other hand, Form 2 is an anti-parallel G-quadruplex comprising two G-tetrads and a G•C•G•C tetrad; the structure has three lateral loops with the middle stem-loop made up of two Watson-Crick G•C base pairs. These structures provide valuable information for the design of G-quadruplex-specific ligands for PIM1 transcription regulation.

Beyond the consensus definition of G-quadruplex-forming motifs with tracts of continuous guanines, G-quadruplexes harboring bulges in the G-tetrad core are prevalent in the human genome. Here, we study the incorporation of a duplex hairpin within a bulge of a G-quadruplex. The NMR solution structure of a G-quadruplex containing a duplex bulge was resolved, revealing the structural details of the junction between the duplex bulge and the G-quadruplex. Unexpectedly, instead of an orthogonal connection the duplex stem was observed to stack below the G-quadruplex forming a unique quadruplex-duplex junction. Breaking up of the immediate base pair step at the junction, coupled with a narrowing of the duplex groove within the context of the bulge, led to a progressive transition between the quadruplex and duplex segments. This study revealed that a duplex bulge can be formed at various positions of a G-quadruplex scaffold. In contrast to a non-structured bulge, the stability of a G-quadruplex slightly increases with an increase in the duplex bulge size. A G-quadruplex structure containing a duplex bulge of up to 33 nt in size was shown to form, which was much larger than the previously reported 7-nt bulge. With G-quadruplexes containing duplex bulges representing new structural motifs with potential biological significance, our findings would broaden the definition of potential G-quadruplex-forming sequences. Nanyang Technological University National Research Foundation (NRF) Published version Singapore National Research Foundation Investigatorship [NRF-NRFI2017-09]; Nanyang Technological University grants (to A.T.P.). Funding for open access charge: Singapore National Research Foundation.

G-rich DNA sequences with tracts of three or more continuous guanines (G≥3) are known to have high propensity to adopt stable G-quadruplex (G4) structures. Bioinformatic analyses suggest high prevalence of G-rich sequences with short G-tracts (G≤2) in the human genome. However, due to limited structural studies, the folding principles of such sequences remain largely unexplored and hence poorly understood. Here, we present the solution NMR structure of a sequence named AT26 consisting of irregularly spaced G2 tracts and two isolated single guanines. The structure is a four-layered G4 featuring two bi-layered blocks, locked between themselves in an unprecedented fashion making it a stable scaffold. In addition to edgewise and propeller-type loops, AT26 also harbors two V-shaped loops: a 2-nt V-shaped loop spanning two G-tetrad layers and a 0-nt V-shaped loop spanning three G-tetrad layers, which are named as VS- and VR-loop respectively, based on their distinct structural features. The intra-lock motif can be a basis for extending the G-tetrad core and a very stable intra-locked G4 can be formed by a sequence with G-tracts of various lengths including several G2 tracts. Findings from this study will aid in understanding the folding of G4 topologies from sequences containing irregularly spaced multiple short G-tracts. Ministry of Education (MOE) Nanyang Technological University National Research Foundation (NRF) Published version Singapore National Research Foundation Investigatorship [NRF-NRFI2017-09]; Singapore Ministry of Education Academic Research Fund Tier 2 [MOE2015-T2-1- 092]; Nanyang Technological University (NTU Singapore) grants (to A.T.P.). Funding for open access charge: Singapore National Research Foundation.

A four-stranded scaffold of nucleic acids termed G-quadruplex (G4) has found growing applications in nano- and biotechnology. Propeller loops are a hallmark of the most stable intramolecular parallel-stranded G4s. To date, propeller loops have been observed to span only a maximum of three G-tetrad layers. Going beyond that would allow creation of more stable scaffolds useful for building robust nanodevices. Here we investigate the formation of propeller loops spanning more than three layers. We showed that native nucleotide sequences are incompatible to-wards this goal, and we identified synthetic non-nucleotide linkers that form propeller loop across four layers. With the established linkers, we constructed a four-layered intramolecular parallel-stranded G4, which exhibited ultra-high thermal stability. Control on loop design would augment the toolbox towards engineering of G4-based nano-scaffolds for diverse applications. National Research Foundation (NRF) Submitted/Accepted version This work was supported by Singapore National Research Foundation Investigatorship (NRF-NRFI2017-09) and Nanyang Technological University grants to A.T.P.

Extracellular DNA, or eDNA, is recognised as a critical biofilm component; however, it is not understood how it forms networked matrix structures. Here, we isolate eDNA from static-culture Pseudomonas aeruginosa biofilms using ionic liquids to preserve its biophysical signatures of fluid viscoelasticity and the temperature dependency of DNA transitions. We describe a loss of eDNA network structure as resulting from a change in nucleic acid conformation, and propose that its ability to form viscoelastic structures is key to its role in building biofilm matrices. Solid-state analysis of isolated eDNA, as a proxy for eDNA structure in biofilms, reveals non-canonical Hoogsteen base pairs, triads or tetrads involving thymine or uracil, and guanine, suggesting that the eDNA forms G-quadruplex structures. These are less abundant in chromosomal DNA and disappear when eDNA undergoes conformation transition. We verify the occurrence of G-quadruplex structures in the extracellular matrix of intact static and flow-cell biofilms of P. aeruginosa, as displayed by the matrix to G-quadruplex-specific antibody binding, and validate the loss of G-quadruplex structures in vivo to occur coincident with the disappearance of eDNA fibres. Given their stability, understanding how extracellular G-quadruplex structures form will elucidate how P. aeruginosa eDNA builds viscoelastic networks, which are a foundational biofilm property.

The intricate arrangement of numerous and closely placed chromophores on nanoscale scaffolds can lead to key photonic applications ranging from optical waveguides and antennas to signal-enhanced fluorescent sensors. In this regard, the self-assembly of dye-appended DNA sequences into programmed photonic architectures is promising. However, the dense-packing of dyes can result in not only compromised DNA assembly (leading to ill-defined structures and precipitates) but also to essentially non-fluorescent systems (due to π-π aggregation). Here, we introduce a two-step “tether and mask” strategy wherein large porphyrin dyes are first attached to short G-quadruplex forming sequences and then reacted with per-O-methylated β-cyclodextrin (PMβCD) caps, to form supramolecular synthons featuring the porphyrin fluor fixed into a masked porphyrin lantern (PL) state, due to intramolecular host-guest interactions in water. The PL-DNA sequences can then be self-assembled into cyclic architectures or unprecedented G-wires tethered with hundreds of porphyrin dyes. Importantly, despite the closely arrayed PL units (~2 nm), the dyes behave as bright chromophores (up to 180-fold brighter than the analogues lacking the PMβCD masks). Since other self-assembling scaffolds, dyes, and host molecules can be used in this modular approach, this manuscript lays a general strategy for the bottom-up aqueous self-assembly of bright nanomaterials containing densely packed dyes.

Recently, we observed the first example of a left‐handed G‐quadruplex structure formed by natural DNA, named Z‐G4 . We analysed the Z‐G4 structure and inspected its primary 28‐nt sequence in order to identify motifs that convey the unique left‐handed twist. Using circular dichroism spectroscopy, NMR spectroscopy, and X‐ray crystallography, we revealed a minimal sequence motif of 12 nt (GTGGTGGTGGTG) for formation of the left‐handed DNA G‐quadruplex, which is found to be highly abundant in the human genome. A systematic analysis of thymine loop mutations revealed a moderate sequence tolerance, which would further broaden the space of sequences prone to left‐handed G‐quadruplex formation. NRF (Natl Research Foundation, S’pore) MOE (Min. of Education, S’pore) Accepted version

Joining peptides and oligonucleotides offers potential benefits, but current methods remain laborious. Here we present a novel approach towards enzymatic ligation of the two modalities through the development of tag phosphoramidites that readily couple onto oligonucleotides. This simple and highly efficient approach paves the way towards streamlined development and production of peptide/protein-oligonucleotide conjugates. Nanyang Technological University National Research Foundation (NRF) Submitted/Accepted version This research was supported by Singapore National Research Foundation (NRF-NRFI2017-09) and Nanyang Technological University.

Cations are critical for the folding and assembly of nucleic acids. In G-quadruplex structures, cations can bind between stacked G-tetrads and coordinate with negatively charged guanine carbonyl oxygens. They usually exchange between binding sites and with the bulk in solution with time constants ranging from sub-millisecond to seconds. Here we report the first observation of extremely long-lived K+ and NH4+ ions, with an exchange time constant on the order of an hour, when coordinated at the center of a left-handed G-quadruplex DNA. A single-base mutation, that switched one half of the structure from left- to right-handed conformation resulting in a right–left hybrid G-quadruplex, was shown to remove this long-lived behaviour of the central cation., An extremely long-lived cation has been detected in left-handed G-quadruplexes.

We generated a novel G-quadruplex (G4)-specific endonuclease by fusing a G4 recognition domain of the RHAU helicase with a cleavage domain of the Fok1 nuclease. The fusion protein can specifically bind a parallel G4 and cleave a double-stranded DNA (dsDNA) next to it. The new endonuclease could be used to detect a G4 in a long dsDNA, providing a useful tool for mapping the formation of G4s in the genome. Ministry of Education (MOE) Accepted version

The discovery of the GFP-type dye DFHBI that becomes fluorescent upon binding to an RNA aptamer, termed Spinach, led to the development of a variety of fluorogenic RNA systems that enable genetic encoding of living cells. In view of increasing interest in small RNA aptamers and the scarcity of their photophysical characterisation, this paper is a model study on Baby Spinach, a truncated Spinach aptamer with half its sequence. Fluorescence and fluorescence excitation spectra of DFHBI complexes of Spinach and Baby Spinach are known to be similar. Surprisingly, a significant divergence between absorption and fluorescence excitation spectra of the DFHBI/RNA complex was observed on conditions of saturation at large excess of RNA over DFHBI. Since absorption spectra were not reported for any Spinach-type aptamer, this effect is new. Quantitative modelling of the absorption spectrum based on competing dark and fluorescent binding sites could explain it. However, following reasoning of fluorescence lifetimes of bound DFHBI, femtosecond-fluorescence lifetime profiles would be more supportive of the notion that the abnormal absorption spectrum is largely caused by trans-isomers formed within the cis-bound DFHBI/RNA complex. Independent of the origin, the unexpected discrepancy between absorption and fluorescence excitation spectra allows for easily accessed screening and insight into the efficiency of a fluorogenic dye/RNA system. Nanyang Technological University National Research Foundation (NRF) Published version This research was supported by the Vietnam National Foundation for Science and Technology Development (NAFOSTED) under grant number 106.02-2018.11 (to Nguyen Thuan Dao), Nanyang Technological University grants to M.-E. Michel-Beyerle and to Anh Tuan Phan. Alexander Voityuk is grateful for financial support from the MINECO, Spanish Ministerio de Economía y Competitividad. This work was supported in part by the Singapore National Research Foundation under its Campus for Research Excellence and Technological Enterprise (CREATE) programme.

G-quadruplex (G4) DNA structures with a left-handed backbone progression have unique and conserved structural features. Studies on sequence dependency of the structures revealed the prerequisites and some minimal motifs required for left-handed G4 formation. To extend the boundaries, we explore the adaptability of left-handed G4s towards the existence of bulges. Here we present two X-ray crystal structures and an NMR solution structure of left-handed G4s accommodating one, two and three bulges. Bulges in left-handed G4s show distinct characteristics as compared to those in right-handed G4s. The elucidation of intricate structural details will help in understanding the possible roles and limitations of these unique structures. Ministry of Education (MOE) Nanyang Technological University National Research Foundation (NRF) Published version Singapore National Research Foundation Investigatorship [NRF-NRFI2017-09]; Singapore Ministry of Education Academic Research Fund Tier 2 [MOE2015-T2- 1092]; Nanyang Technological University (NTU Singapore) grants (to A.T.P.). Funding for open access charge: Nanyang Technological University.

G-quadruplex (G4)-binding proteins regulate important biological processes, but their interaction networks are poorly understood. We report the first use of G4 as warhead of a proteolysis-targeting chimera (G4-PROTAC) for targeted degradation of a G4-binding protein (RHAU/DHX36). G4-PROTAC provides a new way to explore G4-protein networks and to develop potential therapeutics. Ministry of Education (MOE) National Research Foundation (NRF) Submitted/Accepted version This research was supported by Singapore Ministry of Education (MOE) Academic Research Fund Tier 2 (MOE2018-T2-2- 029) and the Singapore National Research Foundation Investigatorship (NRF-NRFI2017-09) to A. T. P.

Tyrosine kinase 2 (TYK2) is a member of the JAK family of nonreceptor tyrosine kinase, together with JAK1, JAK2, and JAK3. JAKs are important signaling mediators of many proinflammatory cytokines and represent compelling pharmacological targets for autoimmune and inflammatory diseases. Pan-acting small-molecule JAK inhibitors were approved for the treatment of rheumatoid arthritis and ulcerative colitis. However, their limited selectivity among JAK members have led to undesirable side effects, driving a search toward specific JAK inhibitors. Recently, TYK2 has emerged as a target of choice for the treatment of autoimmune diseases and severe COVID-19 with an optimum balance between efficacy and safety, based on observations from human genetics studies and clinical outcomes of several agents targeting cytokine pathways for which TYK2 plays an essential role. In this article, we address selective targeting of TYK2 from the genetic sequence space through development of antisense oligonucleotides (ASOs) against TYK2 mRNA. Potent ASO candidates were identified from the screening of over 200 ASOs using locked nucleic acid gapmer design. The lead ASOs exhibited potent and selective knockdown of TYK2 mRNA and protein across a panel of model human cell lines in a dose-dependent manner, showing no reduction in the mRNA and protein expression levels of other JAK paralogs. In agreement with the depletion of TYK2 proteins, several TYK2-mediated cytokine signaling pathways, including IFN-α and IL-12, were inhibited upon ASO treatment. Our results established the TYK2 ASOs as investigational tool compound and potential therapeutic agent for the treatment of autoimmune diseases and severe COVID-19.

Cyclic peptide natural products have served as important drug molecules, with several examples used clinically. Enzymatic or chemical macrocyclization is the key transformation for constructing these chemotypes. Methods to generate new and diverse cyclic peptide scaffolds enabling the modular and predictable synthesis of peptide libraries are desirable in drug discovery platforms. Here we identify a suite of post-translational modifying enzymes from bacteria that install single or multiple strained cyclophane macrocycles. The crosslinking occurs on three-residue motifs that include tryptophan or phenylalanine to form indole- or phenyl-bridged cyclophanes. The macrocycles display restricted rotation of the aromatic ring and induce planar chirality in the asymmetric indole bridge. The biosynthetic gene clusters originate from a broad range of bacteria derived from marine, terrestrial and human microbiomes. Three-residue cyclophane-forming enzymes define a new and significant natural product family and occupy a distinct region in sequence–function space. A series of enzymes that catalyse the formation of strained peptide cyclophanes through a stereospecific C(sp2)–C(sp3) bond have been identified. Crosslinking occurs on three-residue motifs that include tryptophan or phenylalanine to form indole- or phenyl-bridged cyclophanes. These enzymes are widely distributed in nature and represent promising tools for peptide biotechnology.

The folding kinetics is an important parameter affecting the relevance of DNA and RNA G-quadruplex (G4) structures in biological processes. Previous studies of the G4 folding kinetics mainly depended on fast measurement techniques. In previously available examples of G4s with loops up to three residues, the folding kinetics spanning several orders of magnitude has been reported, ranging from milliseconds to over 100 s. It is difficult to systematically and fundamentally understand the effect of multiple parameters, especially the loop properties, on the G4 folding kinetics, as the G4 fold is often altered when the sequence is varied. In this study, judicious choices of multiple parameters allowed us to bring various systems into the measurable window of a simple UV absorption technique. Using a well-controlled parallel-stranded G4 fold, we were able to systematically investigate the effect of seven different parameters of the environment and loop properties (temperature, K+ concentration, ionic strength, co-solute, loop length, loop sequence, and loop structure) on the G4 folding kinetics. We found that structured loops can drive up the G4 folding: for a long loop, the fast folding of a stem loop can guide the G4 folding and accelerate its folding kinetics by several orders of magnitude compared to an unstructured loop counterpart. Ministry of Education (MOE) National Research Foundation (NRF) Accepted version Singapore National Research Foundation Investigatorship (NRF-NRFI2017-09) Singapore Ministry of Education Academic Research Fund Tier 3 (MOE2012-T3-1-001)

G-quadruplex topological diversity is reviewed based on the structural elements: G-tetrads, G-columns and loops. Each of the structural elements has its own individual and relational components, as well as established topological rules. Individual components consist of G-tetrad configurations, G-column continuity and loop types; relational components comprise of G-tetrad stacking orientation, G-column relative directionality and loops incorporating peripheral structures. Furthermore, the notion of canonical G-quadruplexes was discussed, exploring the previous nomenclature of DNA G-quadruplexes and energetic constraints of RNA G-quadruplexes. Finally, selected examples of both canonical and non-canonical DNA and RNA G-quadruplexes were shown to give perspectives toward the future of G-quadruplex structure and diversity.

G-quadruplexes(G4) arenon-canonical nucleicacidstructures with important implications in biology. Based on an a-helical fragment of the RHAU helicase that displays high specificity for parallelstranded G-quadrplexes, herein we demonstrate its head-to-tail cyclization by a high-efficiency ligase. The cyclic peptide exhibits superior stability and binding affinity to a G-quadruplex, and can serve as an excellent investigational tool for chemical biology applications Ministry of Education (MOE) Submitted/Accepted version This work was supported by Singapore Ministry of Education (MOE) Academic Research Fund Tier 2 (MOE2018-T2-2-029) to A. T. P. and MOE Academic Research Fund Tier 3 (MOE2016-T3- 1-003) to J. P. T

AGRO100 (also known as AS1411) is a G-rich oligonucleotide that has long been established as a potent anti-cancer aptamer. However, the structure of AGRO100 remained unresolved, due to the co-existence of multiple different G-quadruplex conformations. We identified a DNA sequence named AT11, derived from AGRO100, which formed a single major G-quadruplex conformation and exhibited a similar anti-proliferative activity as AGRO100. The solution structure of AT11 revealed a four-layer G-quadruplex comprising of two propeller-type parallel-stranded subunits connected through a central linker. The stacking between the two subunits occurs at the 3΄-end of the first block and the 5΄-end of the second block. The structure of the anti-proliferative DNA sequence AT11 will allow greater understanding on the G-quadruplex folding principles and aid in structural optimization of anti-proliferative oligonucleotides. NRF (Natl Research Foundation, S’pore) MOE (Min. of Education, S’pore) Published version

Functionalized nanoparticles have seen valuable applications, particularly in the delivery of therapeutic and diagnostic agents in biological systems. However, the manufacturing of such nano-scale systems with the consistency required for biological application can be challenging, as variation in size and shape have large influences in nanoparticle behavior in vivo. We report on the development of a versatile nano-scaffold based on the modular functionalization of a DNA G-quadruplex. DNA sequences are functionalized in a modular fashion using well-established phosphoramidite chemical synthesis with nucleotides containing modification of the amino (N2) position of the guanine base. In physiological conditions, these sequences fold into well-defined G-quadruplex structures. The resulting DNA nano-scaffolds are thermally stable, consistent in size, and functionalized in a manner that allows for control over the density and relative orientation of functional chemistries on the nano-scaffold surface. Various chemistries including small modifications (N2-methyl-guanine), bulky aromatic modifications (N2-benzyl-guanine), and long chain-like modifications (N2-6-amino-hexyl-guanine) are tested and are found to be generally compatible with G-quadruplex formation. Furthermore, these modifications stabilize the G-quadruplex scaffold by 2.0–13.3 °C per modification in the melting temperature, with concurrent modifications producing extremely stable nano-scaffolds. We demonstrate the potential of this approach by functionalizing nano-scaffolds for use within the biotin–avidin conjugation approach. NRF (Natl Research Foundation, S’pore) MOE (Min. of Education, S’pore) Published version

Cyclic dinucleotides have emerged as important secondary messengers and cell signaling molecules that regulate several cell responses. A guanine-deficit G-quadruplex structure formation by a sequence containing (4n – 1) guanines, n denoting the number of G-tetrad layers, was previously reported. Here, a (4n – 1) G-quadruplex structure is shown to be capable of binding guanine-containing dinucleotides in micromolar affinity. The guanine base of the dinucleotides interacts with a vacant G-triad, forming four additional Hoogsteen hydrogen bonds to complete a G-tetrad. Solution structures of two complexes, both comprised of a (4n – 1) G-quadruplex structure, one bound to a linear dinucleotide (d(AG)) and the other to a cyclic dinucleotide (cGAMP), are solved using NMR spectroscopy. The latter suggests sufficiently strong interaction between the guanine base of the dinucleotide and the vacant G-triad, which acts as an anchor point of binding. The binding interfaces from the two solution structures provide useful information for specific ligand design. The results also infer that other guanine-containing metabolites of a similar size have the capability of binding G-quadruplexes, potentially affecting the expression of the metabolites and functionality of the bound G-quadruplexes. NRF (Natl Research Foundation, S’pore) Accepted version

A knot‐like G‐quadruplex peripheral structure is formed by a 7‐nt DNA sequence DL7 (TGTTGGT), in which six out of its seven nucleobases participate in compact base‐pairing interactions. Here, the solution NMR structure of a 24‐nt DNA oligonucleotide containing the DL7 sequence shows the interaction between a two‐layer anti‐parallel G‐quadruplex core and the peripheral knot‐like structure, including the construction of two sharp turns in the DNA backbone. The formation of this novel structural element highlights the intricate properties of single‐stranded DNA folding in presence of G‐quadruplex‐forming motifs. We demonstrated the compatibility of the DL7 knot‐like structure with various G‐quadruplexes, which could have implications in drug design and DNA engineering. NRF (Natl Research Foundation, S’pore) MOE (Min. of Education, S’pore) Accepted version

Analogous to the B- and Z-DNA structures in double-helix DNA, there exist both right- and left-handed quadruple-helix (G-quadruplex) DNA. Numerous conformations of right-handed and a few left-handed G-quadruplexes were previously observed, yet they were always identified separately. Here, we present the NMR solution and X-ray crystal structures of a right- and left-handed hybrid G-quadruplex. The structure reveals a stacking interaction between two G-quadruplex blocks with different helical orientations and displays features of both right- and left-handed G-quadruplexes. An analysis of loop mutations suggests that single-nucleotide loops are preferred or even required for the left-handed G-quadruplex formation. The discovery of a right- and left-handed hybrid G-quadruplex further expands the polymorphism of G-quadruplexes and is potentially useful in designing a left-to-right junction in G-quadruplex engineering. NRF (Natl Research Foundation, S’pore) MOE (Min. of Education, S’pore) Published version

We developed a ribonuclease for site-specific targeting and cleavage of single-stranded RNA. The engineered RNase protein was constructed by incorporating two independent functional domains, an RNase HI domain that could cleave the RNA strand in a DNA-RNA hybrid, and a domain of the RHAU protein that could selectively recognize a parallel DNA G-quadruplex (G4). The newly designed RNase first recruits a DNA guide oligonucleotide containing both a parallel G4 motif and a template sequence complementary to the target RNA. This RNase:DNA complex targets and efficiently cleaves the single-stranded RNA in a site-specific manner. A major cleavage site occurs at the RNA region that is complementary to the DNA template sequence. The newly designed RNase can serve as a simple tool for RNA manipulation and probing RNA structure. NRF (Natl Research Foundation, S’pore) Published version

Poly (ADP-ribose) polymerase 1 (PARP1) has emerged as an attractive target for cancer therapy due to its key role in DNA repair processes. Inhibition of PARP1 in BRCA-mutated cancers has been observed to be clinically beneficial. Recent genome-mapping experiments have identified a non-canonical G-quadruplex-forming sequence containing bulges within the PARP1 promoter. Structural features, like bulges, provide opportunities for selective chemical targeting of the non-canonical G-quadruplex structure within the PARP1 promoter, which could serve as an alternative therapeutic approach for the regulation of PARP1 expression. Here we report the G-quadruplex structure formed by a 23-nucleotide G-rich sequence in the PARP1 promoter. Our study revealed a three-layered intramolecular (3+1) hybrid G-quadruplex scaffold, in which three strands are oriented in one direction and the fourth in the opposite direction. This structure exhibits unique structural features such as an adenine bulge and a G·G·T base triple capping structure formed between the central edgewise loop, propeller loop and 5′ flanking terminal. Given the highly important role of PARP1 in DNA repair and cancer intervention, this structure presents an attractive opportunity to explore the therapeutic potential of PARP1 inhibition via G-quadruplex DNA targeting. NRF (Natl Research Foundation, S’pore) Published version

While extracellular DNA (eDNA) is recognized as a critical biofilm matrix component, it is not understood how it contributes to biofilm function. Here we isolate eDNA from Pseudomonas biofilms using ionic liquids, and discover that its key biophysical signatures, i.e. fluid viscoelasticity, nucleic acid conformation, and temperature and pH dependencies of gel to solution transitions, are maintained. Solid-state analysis of isolated eDNA, as a proxy for eDNA structure in biofilms, revealed non-canonical Hoogsteen base pairs, triads or tetrads involving guanine and thymine or uracil. These were less abundant in chromosomal DNA and undetected as eDNA underwent gel-sol transition. Purine-rich RNA was present in the eDNA network, which potentially enables eDNA to be the main cross-linking exopolymer in the matrix through non-canonical nucleobase interactions. Our study suggests that Pseudomonas assemble extracellular DNA and RNA into a network with viscoelastic properties, which underpin their persistence and spreading, and may aid the development of more effective controls for biofilm-associated infections.

The long terminal repeat (LTR) of the proviral human immunodeficiency virus (HIV)-1 genome is integral to virus transcription and host cell infection. The guanine-rich U3 region within the LTR promoter, previously shown to form G-quadruplex structures, represents an attractive target to inhibit HIV transcription and replication. In this work, we report the structure of a biologically relevant G-quadruplex within the LTR promoter region of HIV-1. The guanine-rich sequence designated LTR-IV forms a well-defined structure in physiological cationic solution. The nuclear magnetic resonance (NMR) structure of this sequence reveals a parallel-stranded G-quadruplex containing a single-nucleotide thymine bulge, which participates in a conserved stacking interaction with a neighboring single-nucleotide adenine loop. Transcription analysis in a HIV-1 replication competent cell indicates that the LTR-IV region may act as a modulator of G-quadruplex formation in the LTR promoter. Consequently, the LTR-IV G-quadruplex structure presented within this work could represent a valuable target for the design of HIV therapeutics.

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.

International audience; The interaction of a macrocyclic tetraoxazole compound, L2H2‐4OTD (1), with two aminoalkyl side chains and telomeric i‐motif, was investigated by means of electrophoretic mobility shift assay, circular dichroism spectroscopy, mass spectrometry and NMR spectroscopy analyses. The results indicate that 1 interacts with the i‐motif structure at two preferred binding sites.

We investigate the self-assembly of short pieces of the Tetrahymena telomeric DNA sequence d[G4T2G4] in physiologically relevant aqueous solution using atomic force microscopy (AFM). Wire-like structures (G-wires) of 3.0 nm height with well-defined surface periodic features were observed. Analysis of high-resolution AFM images allowed their classification based on the periodicity of these features. A major species is identified with periodic features of 4.3 nm displaying left-handed ridges or zigzag features on the molecular surface. A minor species shows primarily left-handed periodic features of 2.2 nm. In addition to 4.3 and 2.2 nm ridges, background features with periodicity of 0.9 nm are also observed. Using molecular modeling and simulation, we identify a molecular structure that can explain both the periodicity and handedness of the major G-wire species. Our results demonstrate the potential structural diversity of G-wire formation and provide valuable insight into the structure of higher-order intermolecular G-quadruplexes. Our results also demonstrate how AFM can be combined with simulation to gain insight into biomolecular structure., DNA and RNA G-quadruplexes can stack to form higher-order structures called G-wires. Here the authors report high-resolution AFM images of higher-order DNA G-quadruplexes in aqueous solution that could impact the design of G-wire based nanodevices and the understanding of G-wires in biology.

A copper complex embedded in the structure of a water-soluble naphthalene diimide has been designed to bind and cleave G-quadruplex DNA. We describe the properties of this ligand, including its catalytic activity in the generation of ROS. FRET melting, CD, NMR, gel sequencing, and mass spectrometry experiments highlight a unique and unexpected selectivity in cleaving G-quadruplex sequences. This selectivity relies both on the binding affinity and structural features of the targeted G-quadruplexes. Published version