A Catalytic and Selective Scissoring Molecular Tool for Quadruplex Nucleic Acids

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

Development and application of NMR spectroscopy in G-quadruplexes : structure, dynamics and formation in biofilm

G-quadruplex is a secondary non-canonical structure of nucleic acid which gained popularity due to increasing evidence of its biological functions, potential therapeutic prospects and nanotechnology applications. Encouraged for a better comprehension of G-quadruplex as an alternative DNA structures, we address multiple angles of G-quadruplex properties including its dynamics, ligand binding kinetics, numerous novel topologies and potential role in biofilm networking. This research was accomplished through a combination of biophysical techniques, primarily Nuclear Magnetic Resonance (NMR) spectroscopy. In this dissertation, G-quadruplex conformational and ligand-interaction dynamics are investigated with NMR spectroscopy. In particular, we developed two specific NMR methodologies for the measurements of the rotation rates of amino protons in guanines of G-quadruplexes via lineshape analysis and the determination of the residence time of ligands bound to G-quadruplex receptors via NOESY exchange analysis. Applications of NMR spectroscopy in the investigation of novel G-quadruplex folding topologies and the analysis of G-quadruplex formation in biofilm are demonstrated. Multiple high-resolution structures are resolved using NMR spectroscopy: (1) Complex structures of 11-guanine G-quadruplex (D4 ) bound to cell-signaling metabolite cyclic guanosine-adenosine monophosphate (cGAMP), (2) a fold-back G-quadruplex structure featuring a triad and base-pair formation on an anti-parallel G-quadruplex core (AT21S ), and (3) a left and right-handed G-quadruplex hybrid formation from multiple trinucleotide repeats (GGT ) 8 . Additionally, we de-termined that extracellular nucleic acid is the main contributor towards polymer networking property of P. aeruginosa biofilm (extracellular matrix). We proved the existence of non-canonical RNA-DNA complexes and potential formation of G-quadruplexes are responsible for the gel-networking characteristic of biofilms. The methodologies presented in this thesis — lineshape analysis and NOESY exchange analysis — contribute as effective options in measuring G-quadruplex dynamics. Furthermore, the applications of NMR spectroscopy explored here provide insights on: (1) Vacancy-bearing G-quadruplex as metabolite binder, (2) fundamentals of G-quadruplex structural elements of fold-back diagonal loops, (3) expansion of G-quadruplex diversity based on the existence of left-handed right-handed junction, and (4) potential biological importance of G-quadruplexes in biofilm network.Doctor of Philosoph

Solution structures of a G-quadruplex bound to linear- and cyclic-dinucleotides

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 versio

Rotation of Guanine Amino Groups in G-Quadruplexes: A Probe for Local Structure and Ligand Binding

Nucleic acids are dynamic molecules whose functions may depend on their conformational fluctuations and local motions. In particular, amino groups are dynamic components of nucleic acids that participate in the formation of various secondary structures such as G-quadruplexes. Here, we present a cost-efficient NMR method to quantify the rotational dynamics of guanine amino groups in G-quadruplex nucleic acids. An isolated spectrum of amino protons from a specific tetrad-bound guanine can be extracted from the nuclear Overhauser effect spectroscopy spectrum based on the close proximity between the intra-residue imino and amino protons. We apply the method in different structural contexts of G-quadruplexes and their complexes. Our results highlight the role of stacking and hydrogen-bond interactions in restraining amino-group rotation. The measurement of the rotation rate of individual amino groups could give insight into the dynamic processes occurring at specific locations within G-quadruplex nucleic acids, providing valuable probes for local structure, dynamics, and ligand binding.MOE (Min. of Education, S’pore

An unprecedented knot‐like G‐quadruplex peripheral motif

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 versio

A novel minimal motif for left-handed G-quadruplex formation

International audienceA recent study on the left-handed G-quadruplex (LHG4) DNA revealed a 12-nt minimal motif GTGGTGGTGGTG with the ability to independently form an LHG4 and to drive an adjacent sequence to LHG4 formation. Here we have identified a second LHG4-forming motif, GGTGGTGGTGTG, and determined the X-ray crystal structure of an LHG4 involving this motif. Our structural analysis indicated the role of split guanines and single thymine loops in promoting LHG4 formation

Intra-locked G-quadruplex structures formed by irregular DNA G-rich motifs

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 UniversityNational Research Foundation (NRF)Published versionSingapore 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

Crystal structures of an HIV-1 integrase aptamer: formation of a water-mediated A•G•G•G•G pentad in an interlocked G-quadruplex

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 versionThis 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)

RNA alternative splicing prediction with discrete compositional energy network

A single gene can encode for different protein versions through a process called alternative splicing. Since proteins play major roles in cellular functions, aberrant splicing profiles can result in a variety of diseases, including cancers. Alternative splicing is determined by the gene's primary sequence and other regulatory factors such as RNA-binding protein levels. With these as input, we formulate the prediction of RNA splicing as a regression task and build a new training dataset (CAPD) to benchmark learned models. We propose discrete compositional energy network (DCEN) which leverages the hierarchical relationships between splice sites, junctions and transcripts to approach this task. In the case of alternative splicing prediction, DCEN models mRNA transcript probabilities through its constituent splice junctions' energy values. These transcript probabilities are subsequently mapped to relative abundance values of key nucleotides and trained with ground-truth experimental measurements. Through our experiments on CAPD1, we show that DCEN outperforms baselines and ablation variants.Nanyang Technological UniversityNational Research Foundation (NRF)Published versionThis work is supported by the Data Science and Artificial Intelligence Research Center (DSAIR), the School of Computer Science and Engineering at Nanyang Technological University and the Singapore National Research Foundation Investigatorship (NRFNRFI2017-09)

Guanine anchoring : a strategy for specific targeting of a G-quadruplex using short PNA, LNA and DNA molecules

Two separate structural elements of a G-quadruplex (G4), a vacant site and a flanking single-strand, provide an opportunity for specific targeting of a particular G4 structure via dual recognition. Here, we show that a short peptide nucleic acid (PNA) can specifically recognize and bind to a G4 at sub-micromolar affinity based on both G-tetrad vacant site filling and complementary duplex formation. This sequence-guided guanine-anchoring strategy can be further developed for specific targeting of G4 structures using short DNA, LNA and PNA strands.NRF (Natl Research Foundation, S’pore)Accepted versio