High-Resolution NMR Structures of Intrastrand Hairpins Formed by CTG Trinucleotide Repeats.

The CAG and CTG trinucleotide repeat expansions cause more than 10 human neurodegenerative diseases. Intrastrand hairpins formed by trinucleotide repeats contribute to repeat expansions, establishing them as potential drug targets. High-resolution structural determination of CAG and CTG hairpins poses as a long-standing goal to aid drug development, yet it has not been realized due to the intrinsic conformational flexibility of repetitive sequences. We herein investigate the solution structures of CTG hairpins using nuclear magnetic resonance (NMR) spectroscopy and found that four CTG repeats with a clamping G-C base pair was able to form a stable hairpin structure. We determine the first solution NMR structure of dG(CTG)4C hairpin and decipher a type I folding geometry of the TGCT tetraloop, wherein the two thymine residues form a T·T loop-closing base pair and the first three loop residues continuously stack. We further reveal that the CTG hairpin can be bound and stabilized by a small-molecule ligand, and the binding interferes with replication of a DNA template containing CTG repeats. Our determined high-resolution structures lay an important foundation for studying molecular interactions between native CTG hairpins and ligands, and benefit drug development for trinucleotide repeat expansion diseases.

Structural investigation of pathogenic RFC1 AAGGG pentanucleotide repeats reveals a role of G-quadruplex in dysregulated gene expression in CANVAS.

An expansion of AAGGG pentanucleotide repeats in the replication factor C subunit 1 (RFC1) gene is the genetic cause of cerebellar ataxia, neuropathy, and vestibular areflexia syndrome (CANVAS), and it also links to several other neurodegenerative diseases including the Parkinson's disease. However, the pathogenic mechanism of RFC1 AAGGG repeat expansion remains enigmatic. Here, we report that the pathogenic RFC1 AAGGG repeats form DNA and RNA parallel G-quadruplex (G4) structures that play a role in impairing biological processes. We determine the first high-resolution nuclear magnetic resonance (NMR) structure of a bimolecular parallel G4 formed by d(AAGGG)2AA and reveal how AAGGG repeats fold into a higher-order structure composed of three G-tetrad layers, and further demonstrate the formation of intramolecular G4s in longer DNA and RNA repeats. The pathogenic AAGGG repeats, but not the nonpathogenic AAAAG repeats, form G4 structures to stall DNA replication and reduce gene expression via impairing the translation process in a repeat-length-dependent manner. Our results provide an unprecedented structural basis for understanding the pathogenic mechanism of AAGGG repeat expansion associated with CANVAS. In addition, the high-resolution structures resolved in this study will facilitate rational design of small-molecule ligands and helicases targeting G4s formed by AAGGG repeats for therapeutic interventions.

Structures and conformational dynamics of DNA minidumbbells in pyrimidine-rich repeats associated with neurodegenerative diseases.

Expansions of short tandem repeats (STRs) are associated with approximately 50 human neurodegenerative diseases. These pathogenic STRs are prone to form non-B DNA structure, which has been considered as one of the causative factors for repeat expansions. Minidumbbell (MDB) is a relatively new type of non-B DNA structure formed by pyrimidine-rich STRs. An MDB is composed of two tetraloops or pentaloops, exhibiting a highly compact conformation with extensive loop-loop interactions. The MDB structures have been found to form in CCTG tetranucleotide repeats associated with myotonic dystrophy type 2, ATTCT pentanucleotide repeats associated with spinocerebellar ataxia type 10, and the recently discovered ATTTT/ATTTC repeats associated with spinocerebellar ataxia type 37 and familial adult myoclonic epilepsy. In this review, we first introduce the structures and conformational dynamics of MDBs with a focus on the high-resolution structural information determined by nuclear magnetic resonance spectroscopy. Then we discuss the effects of sequence context, chemical environment, and nucleobase modification on the structure and thermostability of MDBs. Finally, we provide perspectives on further explorations of sequence criteria and biological functions of MDBs.

Controlled Self-Assembly of the Catalytic Core of Hydrolases Using DNA Scaffolds.

Precisely organizing functional molecules of the catalytic cores in natural enzymes to promote catalytic performance is a challenging goal in respect to artificial enzyme construction. In this work, we report a DNA-scaffolded mimicry of the catalytic cores of hydrolases, which showed a controllable and hierarchical acceleration of the hydrolysis of fluorescein diacetate (FDA). The results revealed that the efficiency of hydrolysis was greatly increased by the DNA-scaffold-induced proximity of catalytic amino acid residues (histidine and arginine) with up to 4-fold improvement relative to the free amino acids. In addition, DNA-scaffolded one-dimensional and two-dimensional assemblies of multiple catalytic cores could further accelerate the hydrolysis. This work demonstrated that the DNA-guided assembly could be used as a promising platform to build enzyme mimics in a programmable and hierarchical way.

Solution Nuclear Magnetic Resonance Structures of ATTTT and ATTTC Pentanucleotide Repeats Associated with SCA37 and FAMEs.

Expansions of ATTTT and ATTTC pentanucleotide repeats in the human genome are recently found to be associated with at least seven neurodegenerative diseases, including spinocerebellar ataxia type 37 (SCA37) and familial adult myoclonic epilepsy (FAME) types 1, 2, 3, 4, 6, and 7. The formation of non-B DNA structures during some biological processes is thought as a causative factor for repeat expansions. Yet, the structural basis for these pyrimidine-rich ATTTT and ATTTC repeat expansions remains elusive. In this study, we investigated the solution structures of ATTTT and ATTTC repeats using nuclear magnetic resonance spectroscopy. Here, we reveal that ATTTT and ATTTC repeats can form a highly compact minidumbbell structure at the 5'-end using their first two repeats. The high-resolution structure of two ATTTT repeats was determined, showing a regular TTTTA pentaloop and a quasi TTTT/A pentaloop. Furthermore, the minidumbbell structure could escape from proofreading by the Klenow fragment of DNA polymerase I when it was located at five or more base pairs away from the priming site, leading to a small-scale repeat expansion. Results of this work improve our understanding of ATTTT and ATTTC repeat expansions in SCA37 and FAMEs, and provide high-resolution structural information for rational drug design.

NMR solution structures of d(GGCCTG)n repeats associated with spinocerebellar ataxia type 36.

Expansion of d(GGCCTG)n hexanucleotide repeats in the NOP56 gene is the genetic cause of spinocerebellar ataxia type 36 (SCA36) which is an inheritable neurodegenerative disease. Non-B DNA is known to be the structural intermediate causing repeat expansions. Yet, the structure and mechanism of genetic instability of d(GGCCTG)n repeats remain elusive. In this work, we investigated the solution structures of sequences containing two to eight GGCCTG repeats using nuclear magnetic resonance (NMR) spectroscopy. They were found to form diverse secondary structures, including hairpin, duplex and G-quadruplex (G4). Intriguingly, the hairpin structure was present in all the investigated sequences. The NMR solution structure of the hairpin formed by d(GGCCTG)2 was determined, disclosing an unprecedented CCTGGG hexanucleotide loop in which the first and sixth loop residues formed a Watson-Crick loop-closing base pair, the second and third loop residues stacked in the major groove, whereas the fourth and fifth loop residues formed a G·G mismatch. Apart from the hairpin, antiparallel G4 and palindromic duplex structures were found to form in d(GGCCTG)2 and d(GGCCTG)3-8, respectively. Results of this work provide new insights into the genetic instability of d(GGCCTG)n repeats and structure-based drug design for SCA36.

A pH and Mg2+-Responsive Molecular Switch Based on a Stable DNA Minidumbbell Bearing 5' and 3'-Overhangs.

Minidumbbell (MDB) is a non-B DNA structure of which the thermodynamic stability is sensitive to a chemical environment such as pH, serving as a potential structural motif in constructing DNA-based molecular switches. This work aims to design thermodynamically stable MDB structures bearing 5' and 3'-overhanging deoxyribonucleotides in order to examine the possibility of MDB to be functionalized. Via making use of 5-methylcytosine and adjusting the pH of solution to be acidic, MDBs bearing 1-nucleotide (nt) or 2-nt overhanging residues at the 5' and 3'-ends have been obtained. Based on one of the new MDB sequences, we have designed a molecular switch that could respond to dual inputs of pH and Mg2+. The MDB strand and its partner strand formed a duplex (the "ON" state) upon inputting pH 7 and Mg2+, whereas the duplex dissociated to restore the MDB structure (the "OFF" state) upon inputting pH 5 and EDTA. The demonstration on the ability of MDB to sustain 5' and 3'-overhanging residues and the construction of a pH and Mg2+-responsive molecular switch will extend the application of MDB structures in dynamic DNA nanotechnology.

Effects of Adenine Methylation on the Structure and Thermodynamic Stability of a DNA Minidumbbell.

DNA methylation is a prevalent regulatory modification in prokaryotes and eukaryotes. N1-methyladenine (m1A) and N6-methyladenine (m6A) have been found to be capable of altering DNA structures via disturbing Watson-Crick base pairing. However, little has been known about their influences on non-B DNA structures, which are associated with genetic instabilities. In this work, we investigated the effects of m1A and m6A on both the structure and thermodynamic stability of a newly reported DNA minidumbbell formed by two TTTA tetranucleotide repeats. As revealed by the results of nuclear magnetic resonance spectroscopic studies, both m1A and m6A favored the formation of a T·m1A and T·m6A Hoogsteen base pair, respectively. More intriguingly, the m1A and m6A modifications brought about stabilization and destabilization effects on the DNA minidumbbell, respectively. This work provides new biophysical insights into the effects of adenine methylation on the structure and thermodynamic stability of DNA.

5-Methylcytosine Substantially Enhances the Thermal Stability of DNA Minidumbbells.

Minidumbbell (MDB) is a recently identified non-B DNA structure that has been proposed to associate with genetic instabilities. It also serves as a functional structural motif in DNA nanotechnology. DNA molecular switches constructed using MDBs show instant and complete structural conversions with easy manipulations. The availability of stable MDBs can broaden their applications. In this work, we found that substitutions of cytosine with 5-methylcytosine could lead to a significant enhancement in the thermal stabilities of MDBs. Consecutive methylations of cytosine in MDBs brought about cumulative stabilization with a drastic increase in the melting temperature by 23 °C. NMR solution structures of two MDBs containing 5-methylcytosine residues have been successfully determined and revealed that the enhanced stabilities resulted primarily from favorable hydrophobic contacts, more stable base pairs and enhanced base-base stackings involving the methyl group of 5-methylcytosine.

Rational design of a reversible Mg2+/EDTA-controlled molecular switch based on a DNA minidumbbell.

Here we report that incorporation of an abasic site to DNA minidumbbells formed by natural sequences can lead to significant enhancements in their thermodynamic stability. Based on these stable minidumbbells, the first metal ion-controlled molecular switch which can regulate instant and reversible DNA duplex formation and dissociation has been constructed.

Synthesis, characterization, photophysics, and anion binding properties of platinum(ii) acetylide complexes with urea group.

A new class of platinum(ii) acetylide complexes with urea group, [Pt((t)Bu3tpy)(C[triple bond, length as m-dash]CC6H4-4-NHC(O)NHC6H4-4-R)](OTf) ((t)Bu3tpy = 4,4',4''-tri-tert-butyl-2,2':6',2''-terpyridine; R = H (), Cl (), CF3 (), and NO2 ()), has been synthesized and characterized. The crystal structures of , ·DMF·THF, ·CH3CN, and ·CH3CN have been determined by X-ray diffraction. Upon excitation at λ > 380 nm, the solid samples of complexes show orange light at 298 K. The anion binding properties of complexes have been studied by UV-vis titration experiments in CH3CN and DMSO. In general, the log K values of with the same anion in CH3CN depend on the substituent R on the acetylide ligand of and follow this order: R = NO2 () > CF3 () > Cl () > H (). For the same complex with different anions, the log K values are in the following order: F(-) > OAc(-) > Cl(-) > Br(-) ≈ HSO4(-) ≈ NO3(-) > I(-), which is in accordance with the decrease in the basicity of anions. Complex with NO2 group shows a dramatic colour change towards F(-) in DMSO, allowing the naked eye detection of F(-).