CAG RNAs induce DNA damage and apoptosis by silencing NUDT16 expression in polyglutamine degeneration.

DNA damage plays a central role in the cellular pathogenesis of polyglutamine (polyQ) diseases, including Huntington's disease (HD). In this study, we showed that the expression of untranslatable expanded CAG RNA per se induced the cellular DNA damage response pathway. By means of RNA sequencing (RNA-seq), we found that expression of the Nudix hydrolase 16 (NUDT16) gene was down-regulated in mutant CAG RNA-expressing cells. The loss of NUDT16 function results in a misincorporation of damaging nucleotides into DNAs and leads to DNA damage. We showed that small CAG (sCAG) RNAs, species generated from expanded CAG transcripts, hybridize with CUG-containing NUDT16 mRNA and form a CAG-CUG RNA heteroduplex, resulting in gene silencing of NUDT16 and leading to the DNA damage and cellular apoptosis. These results were further validated using expanded CAG RNA-expressing mouse primary neurons and in vivo R6/2 HD transgenic mice. Moreover, we identified a bisamidinium compound, DB213, that interacts specifically with the major groove of the CAG RNA homoduplex and disfavors the CAG-CUG heteroduplex formation. This action subsequently mitigated RNA-induced silencing complex (RISC)-dependent NUDT16 silencing in both in vitro cell and in vivo mouse disease models. After DB213 treatment, DNA damage, apoptosis, and locomotor defects were rescued in HD mice. This work establishes NUDT16 deficiency by CAG repeat RNAs as a pathogenic mechanism of polyQ diseases and as a potential therapeutic direction for HD and other polyQ diseases.

Minidumbbell structures formed by ATTCT pentanucleotide repeats in spinocerebellar ataxia type 10.

Spinocerebellar ataxia type 10 (SCA10) is a progressive genetic disorder caused by ATTCT pentanucleotide repeat expansions in intron 9 of the ATXN10 gene. ATTCT repeats have been reported to form unwound secondary structures which are likely linked to large-scale repeat expansions. In this study, we performed high-resolution nuclear magnetic resonance spectroscopic investigations on DNA sequences containing two to five ATTCT repeats. Strikingly, we found the first two repeats of all these sequences well folded into highly compact minidumbbell (MDB) structures. The 3D solution structure of the sequence containing two ATTCT repeats was successfully determined, revealing the MDB comprises a regular TTCTA and a quasi TTCT/A pentaloops with extensive stabilizing loop-loop interactions. We further carried out in vitro primer extension assays to examine if the MDB formed in the primer could escape from the proofreading function of DNA polymerase. Results showed that when the MDB was formed at 5-bp or farther away from the priming site, it was able to escape from the proofreading by Klenow fragment of DNA polymerase I and thus retained in the primer. The intriguing structural findings bring about new insights into the origin of genetic instability in SCA10.

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.

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.

Achilles Tendon Xanthomas: Fat-Water Separation at Baseline and after Treatment.

Purpose To investigate the fat-water content of Achilles tendon xanthomas at baseline and after treatment and to compare this assessment with that of ultrasonography (US) and other magnetic resonance (MR) imaging-based parameters. Materials and Methods Forty-eight Achilles tendons with clinically apparent xanthomas in 24 patients with familial hypercholesterolemia (FH) (six men, 18 women; mean age ± standard deviation, 58 years ± 9) were compared with 20 Achilles tendons in 10 control subjects without FH (two men, eight women; mean age, 62 years ± 7). US imaging measurements (thickness, width, cross-sectional area, echogenicity) and 3.0-T MR imaging measurements (thickness, width, cross-sectional area, volume, and fat-water separation) of the Achilles tendons were obtained at baseline and in patients with FH at 3 and 6 months after treatment with probucol, a cholesterol-lowering agent. Nonparametric tests compared baseline data, whereas repeated-measures analyses assessed treatment change. Results At baseline, all US and MR imaging-based parameters were higher in xanthoma tendons compared with those in control tendons (all P < .05). The mean relative water content per unit volume was 71% higher (42.0% ± 6.7) in xanthoma tendons than in control tendons (24.5% 6 5.8; P < .001). After 6 months of cholesterol-lowering treatment, only MR imaging measurements of tendon volume (P = .007), relative fat (P = .041), and relative water content (P < .001) showed significant changes. As relative tendon fat content decreased with treatment, relative water content increased. Conclusion Most of the enlargement of Achilles tendon xanthomas is due to an increase in water content rather than fat. For depicting treatment change, relative tendon water content was the most sensitive parameter, followed by tendon volume and relative tendon fat content. © RSNA, 2017 Online supplemental material is available for this article.

Crosslinking reactions of 4-amino-6-oxo-2-vinylpyrimidine with guanine derivatives and structural analysis of the adducts.

DNA interstrand crosslinks (ICLs) are the primary mechanism for the cytotoxic activity of many clinical anticancer drugs, and numerous strategies for forming ICLs have been developed. One such method is using crosslink-forming oligonucleotides (CFOs). In this study, we designed a 4-amino-6-oxo-2-vinylpyrimidine (AOVP) derivative with an acyclic spacer to react selectively with guanine. The AOVP CFO exhibited selective crosslinking reactivity with guanine and thymine in DNA, and with guanine in RNA. These crosslinking reactions with guanine were accelerated in the presence of CoCl2, NiCl2, ZnCl2 and MnCl2. In addition, we demonstrated that the AOVP CFO was reactive toward 8-oxoguanine opposite AOVP in the duplex DNA. The structural analysis of each guanine and 8-oxoguanine adduct in the duplex DNA was investigated by high-resolution NMR. The results suggested that AOVP reacts at the N2 amine in guanine and at the N1 or N2 amines in 8-oxoguanine in the duplex DNA. This study demonstrated the first direct determination of the adduct structure in duplex DNA without enzyme digestion.

Minidumbbell: A New Form of Native DNA Structure.

The non-B DNA structures formed by short tandem repeats on the nascent strand during DNA replication have been proposed to be the structural intermediates that lead to repeat expansion mutations. Tetranucleotide TTTA and CCTG repeat expansions have been known to cause reduction in biofilm formation in Staphylococcus aureus and myotonic dystrophy type 2 in human, respectively. In this study, we report the first three-dimensional minidumbbell (MDB) structure formed by natural DNA sequences containing two TTTA or CCTG repeats. The formation of MDB provides possible pathways for strand slippage to occur, which ultimately leads to repair escape and thus expansion mutations. Our result here shows that MDB is a highly compact structure composed of two type II loops. In addition to the typical stabilizing interactions in type II loops, MDB shows extensive stabilizing forces between the two loops, including two distinctive modes of interactions between the minor groove residues. The formation of MDB enriches the structural diversity of natural DNA sequences, reveals the importance of loop-loop interactions in unusual DNA structures, and provides insights into novel mechanistic pathways of DNA repeat expansion mutations.

The origin of genetic instability in CCTG repeats.

CCTG tetranucleotide repeat expansion is associated with a hereditary neurological disease called myotonic dystrophy type 2 (DM2). The underlying reasons that lead to genetic instability and thus repeat expansion during DNA replication remains elusive. Here, we have shown CCTG repeats have a high propensity to form metastable hairpin and dumbbell structures using high-resolution nuclear magnetic resonance (NMR) spectroscopy. When the repeat length is equal to three, a hairpin with a two-residue CT loop is formed. In addition to the hairpin, a dumbbell structure with two CT-loops is formed when the repeat length is equal to four. Nuclear Overhauser effect (NOE) and chemical shift data reveal both the hairpin and dumbbell structures contain a flexible stem comprising a C-bulge and a T·T mismatch. With the aid of single-site mutation samples, NMR results show these peculiar structures undergo dynamic conformational exchange. In addition to the intrinsic flexibility in the stem region of these structures, the exchange process also serves as an origin of genetic instability that leads to repeat expansion during DNA replication. The structural features provide important drug target information for developing therapeutics to inhibit the expansion process and thus the onset of DM2.

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.

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.

A purine and a backbone discontinuous site alter the structure and thermal stability of DNA minidumbbells containing two pentaloops.

Minidumbbell (MDB) is a noncanonical DNA structure found to form in several pyrimidine-rich short tandem repeats associated with neurodegenerative diseases. The most recently reported MDB contains two pentaloops formed by ATTCT repeats. Here, we studied the effects of a purine residue and a backbone discontinuous site on the structure and thermal stability of MDBs containing two pentaloops. It was found that a purine as the fourth loop residue improved the thermal stability of MDBs containing two regular pentaloops, while a backbone discontinuous site between the third and fourth, or between the fourth and fifth loop residues enhanced the thermal stability of MDBs containing a regular and a quasi pentaloops. The results of this study provide new insights into the sequence criteria and structural basis of MDBs.

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.

Unprecedented hydrophobic stabilizations from a reverse wobble T·T mispair in DNA minidumbbell.

Minidumbbell (MDB) is a newly found non-B DNA structure formed by short single-strand sequences. Up to now, three MDBs have been reported to form at neutral pH by sequences containing two repeats of TTTA, CCTG and CTTG. Among them, the thermodynamically less stable TTTA and CCTG MDBs have been proposed to be the structural intermediates that cause TTTA and CCTG repeat expansions during DNA replication in Staphylococcus aureus pathogen and myotonic dystrophy type 2 patients, respectively. Although the CTTG MDB has a melting temperature of at least 13 °C higher than those of the other two, no CTTG repeat expansion has ever been reported in any genomes. In this study, we successfully determined the solution structure of the CTTG MDB and observed for the first time the formation of a reverse wobble T·T mispair with two symmetric hydrogen bonds. More importantly, we identified unprecedented hydrophobic interactions between the two methyl groups of this T·T mispair and the four 2'-methylene groups of their nearby loop-closing base pair residues. These stabilizations account for the substantial increase in the MDB thermodynamic stability which may govern the occurrence of repeat expansions.Communicated by Ramaswamy H. Sarma [Formula: see text].

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.

High-Resolution Structures of DNA Minidumbbells Comprising Type II Tetraloops with a Purine Minor Groove Residue.

Minidumbbell (MDB) is a newly discovered DNA structure formed by native sequences, which serves as a possible structural intermediate causing repeat expansion mutations in the genome and also a functional structural motif in constructing DNA-based molecular switches. Until now, all the reported MDBs containing two adjacent type II tetraloops were formed by pyrimidine-rich sequences 5'-YYYR YYYR-3' (Y and R represent pyrimidine and purine, respectively), wherein the second and sixth residues folded into the minor groove and interacted with each other. In this study, we have conducted a high-resolution nuclear magnetic resonance (NMR) spectroscopic investigation on alternative MDB-forming sequences and discovered that an MDB could also be formed stably with a purine in the minor groove, which has never been observed in any previously reported DNA type II tetraloops. Our refined NMR solution structures of the two MDBs formed by 5'-CTTG CATG-3' and 5'-CTTG CGTG-3' reveal that the sixth purine residue was driven into the minor groove via base-base stacking with the second thymine residue and adenine stacked better than guanine. The results of our present research work expand the sequence criteria for the formation of MDBs and shed light to explore the significance of MDBs.

NMR investigation of DNA primer-template models: guanine templates are less prone to strand slippage upon misincorporation.

Misaligned structures can result from strand slippage during DNA replication and, if not repaired, would lead to mutations. Previously, we showed that strand slippage can occur upon misincorporation of a dNTP opposite thymine and cytosine templates, resulting in a misaligned structure with a T- or C-bulge. The formation propensity for misaligned structures was found to depend on the type of terminal base pair. In this study, we performed NMR investigations on primer-template models containing a guanine template. Our results reveal guanine templates are less prone to strand slippage than pyrimidine templates. Misalignment was found to occur only in 5'-CG templates with a downstream purine. In addition to the significance of terminal base pair and upstream nucleotide, the present study reveals the importance of the templating base and its downstream nucleotide, which also determine the propensity of strand slippage in primer-templates.

DSHIFT: a web server for predicting DNA chemical shifts.

DSHIFT is a web server for predicting chemical shifts of DNA sequences in random coil form or double helical B-form. The prediction methods are based on sets of published reference chemical shift values and correction factors which account for shielding or deshielding effects from neighboring nucleotides. Proton, carbon and phosphorus chemical shift predictions are available for random coil DNAs. For double helical B-DNA, only proton chemical shift prediction is available. Results from these predictions will be useful for facilitating NMR resonance assignments and investigating structural features of solution DNA molecules. The URL of this server is: http://www.chem.cuhk.edu.hk/DSHIFT.

Effect of 1-methyladenine on double-helical DNA structures.

Methylation at the N1 site of adenine leads to the formation of cytotoxic 1-methyladenine (m1A). Since the N1 site of adenine is involved in the hydrogen bonding of T.A and A.T Watson-Crick base pairs, it is expected that the pairing interactions will be disrupted upon 1-methylation. In this study, high-resolution NMR investigations were performed to determine the effect of m1A on double-helical DNA structures. Interestingly, instead of disrupting hydrogen bonding, we found that 1-methylation altered the T.A Watson-Crick base pair to T(anti).m1A(syn) Hoogsteen base pair, providing insights into the observed differences in AlkB-repair efficiency between dsDNA and ssDNA.

Proton chemical shift prediction of A.A mismatches in B-DNA duplexes.

A proton chemical shift prediction method has been developed for double helical DNAs containing A.A mismatches. This method makes use of the chemical shift prediction scheme for normal B-DNA duplexes developed by Altona and co-workers and a set of A.A mismatch triplet chemical shift values and corrections factors extracted from reference sequences. The triplet values are used for predicting chemical shifts of A.A mismatches whereas the normal B-DNA chemical shifts and correction factors are used for the flanking residues of A.A mismatches. Both 5'- and 3'-correction factors have been determined from the chemical shift differences upon replacing the A.A mismatch in a duplex with an A.T base pair. Based on 560 sets of predicted and experimental chemical shifts, the overall prediction accuracy for various types of protons has been determined to be 0.07 ppm with an excellent correlation coefficient of 0.9996.