Agnieszka Kiliszek

Atomic resolution structure of CAG RNA repeats: structural insights and implications for the trinucleotide repeat expansion diseases

CAG repeats occur predominantly in the coding regions of human genes, which suggests their functional importance. In some genes, these sequences can undergo pathogenic expansions leading to neurodegenerative polyglutamine (poly-Q) diseases. The mutant transcripts containing expanded CAG repeats possibly contribute to pathogenesis in addition to the well-known pathogenic effects of mutant proteins. We have analysed two crystal forms of RNA duplexes containing CAG repeats: (GGCAGCAGCC)2. One of the structures has been determined at atomic resolution (0.95 Å) and the other at 1.9 Å. The duplexes include non-canonical A–A pairs that fit remarkably well within a regular A-helix. All the adenosines are in the anti-conformation and the only interaction within each A–A pair is a single C2-H2···N1 hydrogen bond. Both adenosines in each A–A pair are shifted towards the major groove, although to different extents; the A which is the H-bond donor stands out more (the ‘thumbs-up’ conformation). The main effect on the helix conformation is a local unwinding. The CAG repeats and the previously examined CUG structures share a similar pattern of electrostatic charge distribution in the minor groove, which could explain their affinity for the pathogenesis-related MBNL1 protein.

Structural insights into CUG repeats containing the ‘stretched U–U wobble’: implications for myotonic dystrophy

Tracks containing CUG repeats are abundant in human gene transcripts. Their biological role includes modulation of pre-mRNA splicing, mRNA transport and regulation of translation. Expanded forms of CUG runs are associated with pathogenesis of several neurodegenerative diseases, including myotonic dystrophy type 1. We have analysed two crystal structures of RNA duplexes containing the CUG repeats: G(CUG)2C and (CUG)6. The first of the structures, analysed at 1.23 Å resolution, is of an oligomer designed by us. The second model was obtained after ‘detwinning’ the 1.58 Å X-ray data previously deposited in the PDB. The RNA duplexes are in the A-form in which all the C–G pairs form Watson–Crick interactions while all the uridine pairs can be described as U•U cis wobble having only one hydrogen bond between the bases. The residue, which accepts the H-bond, is inclined towards the minor groove. This previously unreported base pairing can be described as ‘stretched U–U wobble’. The regular hydrogen-bonding pattern of interactions with the solvent, the electrostatic charge distribution and surface features indicate the ligand binding potential of the CUG tracks.

Crystal structures of CGG RNA repeats with implications for fragile X-associated tremor ataxia syndrome

The CGG repeats are present in the 5′-untranslated region (5′-UTR) of the fragile X mental retardation gene FMR1 and are associated with two diseases: fragile X-associated tremor ataxia syndrome (FXTAS) and fragile X syndrome (FXS). FXTAS occurs when the number of repeats is 55–200 and FXS develops when the number exceeds 200. FXTAS is an RNA-mediated disease in which the expanded CGG tracts form stable structures and sequester important RNA binding proteins. We obtained and analysed three crystal structures of double-helical CGG repeats involving unmodified and 8-Br modified guanosine residues. Despite the presence of the non-canonical base pairs, the helices retain an A-form. In the G–G pairs one guanosine is always in the syn conformation, the other is anti. There are two hydrogen bonds between the Watson–Crick edge of G(anti) and the Hoogsteen edge of G(syn): O6·N1H and N7·N2H. The G(syn)-G(anti) pair shows affinity for binding ions in the major groove. G(syn) causes local unwinding of the helix, compensated elsewhere along the duplex. CGG helical structures appear relatively stable compared with CAG and CUG tracts. This could be an important factor in the RNA’s ligand binding affinity and specificity.

Crystallographic characterization of CCG repeats.

CCG repeats are highly over-represented in exons of the human genome. Usually they are located in the 5′ UTR but are also abundant in translated sequences. The CCG repeats are associated with three tri-nucleotide repeat disorders: Huntington’s disease, myotonic dystrophy type 1 and chromosome X-linked mental retardation (FRAXE). In this study, we present two crystal structures containing double-stranded CCG repeats: one of an RNA in the native form, and one containing LNA nucleotides. Both duplexes form A-helices but with strands slipped in the 5′ (native structure) or the 3′ direction (LNA-containing structure). As a result, one of two expected C-C pairs is eliminated from the duplex. Each of the three observed C-C pairs interacts differently, forming either one weak H-bond or none. LNA nucleotides have no apparent effect on the helical parameters but the base stacking is increased compared to the native duplex and the distribution of electrostatic potential in the major groove is changed. The CCG crystal structures explain the thermodynamic fragility of CCG runs and throw light on the observation that the MBNL1 protein recognises CCG runs, as well as CUG and CAG, but not the relatively stable CGG repeats.

Structural studies of CNG repeats

CNG repeats (where N denotes one of the four natural nucleotides) are abundant in the human genome. Their tendency to undergo expansion can lead to hereditary diseases known as TREDs (trinucleotide repeat expansion disorders). The toxic factor can be protein, if the abnormal gene is expressed, or the gene transcript, or both. The gene transcripts have attracted much attention in the biomedical community, but their molecular structures have only recently been investigated. Model RNA molecules comprising CNG repeats fold into long hairpins whose stems generally conform to an A-type helix, in which the non-canonical N-N pairs are flanked by C-G and G-C pairs. Each homobasic pair is accommodated in the helical context in a unique manner, with consequences for the local helical parameters, solvent structure, electrostatic potential and potential to interact with ligands. The detailed three-dimensional profiles of RNA CNG repeats can be used in screening of compound libraries for potential therapeutics and in structure-based drug design. Here is a brief survey of the CNG structures published to date.

Nucleoside bearing boron clusters and their phosphoramidites – building blocks for modified oligonucleotide synthesis

This paper describes a general method for the synthesis of four canonical nucleosides T, dC, dA and dG and their phosphoramidites suitable for automated synthesis of DNA modified with a carborane cage. A boron cluster in the form of an electroneutral, lipophilic 1,2-dicarba-closo-dodecaborane (C2B9H11) or negatively charged, redox-active 7,8-dicarba-nido-undecaborate ion (C2B9H12(−1)) was used as a modifying unit. The method is based on the “click chemistry” type Huisgen–Sharpless–Meldal reaction. All boron cluster-nucleoside conjugates have been characterized electrochemically; they have shown different redox potentials allowing for selective electrochemical identification of individual nucleosides in the mixture. There is also the first description of the crystallographic structure of the boron cluster-nucleoside conjugate: N3-{[(o-carboran-1-yl)propyl]-1N-1,2,3-triazol-4-yl}methylenethymidine.

The first crystal structures of RNA-PNA duplexes and a PNA-PNA duplex containing mismatches-toward anti-sense therapy against TREDs.

PNA is a promising molecule for antisense therapy of trinucleotide repeat disorders. We present the first crystal structures of RNA–PNA duplexes. They contain CUG repeats, relevant to myotonic dystrophy type I, and CAG repeats associated with poly-glutamine diseases. We also report the first PNA–PNA duplex containing mismatches. A comparison of the PNA homoduplex and the PNA–RNA heteroduplexes reveals PNAs intrinsic structural properties, shedding light on its reported sequence selectivity or intolerance of mismatches when it interacts with nucleic acids. PNA has a much lower helical twist than RNA and the resulting duplex has an intermediate conformation. PNA retains its overall conformation while locally there is much disorder, especially peptide bond flipping. In addition to the Watson–Crick pairing, the structures contain interesting interactions between the RNAs phosphate groups and the Π electrons of the peptide bonds in PNA.

Structures of RNA repeats associated with neurological diseases

All RNA molecules possess a ‘propensity’ to fold into complex secondary and tertiary structures. Although they are composed of only four types of nucleotides, they show an enormous structural richness which reflects their diverse functions in the cell. However, in some cases the folding of RNA can have deleterious consequences. Aberrantly expanded, repeated RNA sequences can exhibit gain‐of‐function abnormalities and become pathogenic, giving rise to many incurable neurological diseases. Most RNA repeats form long hairpin structures whose stem consists of noncanonical base pairs interspersed among Watson–Crick pairs. The expanded hairpins have an ability to sequester important proteins and form insoluble nuclear foci. The RNA pathology, common to many repeat disorders, has drawn attention to the structures of the RNA repeats. In this review, we summarize secondary structure probing and crystallographic studies of disease‐related RNA repeat sequences. We discuss the unique structural features which can contribute to the pathogenic properties of the repeated runs. In addition, we present the newest reports concerning structural data linked to therapeutic approaches. WIREs RNA 2017, 8:e1412. doi: 10.1002/wrna.1412

Stabilization of RNA hairpins using non-nucleotide linkers and circularization.

Abstract An RNA hairpin is an essential structural element of RNA. Hairpins play crucial roles in gene expression and intermolecular recognition but are also involved in the pathogenesis of some congenital diseases. Structural studies of the hairpin motifs are impeded by their thermodynamic instability, as they tend to unfold to form duplexes, especially at high concentrations required for crystallography or nuclear magnetic resonance spectroscopy. We have elaborated techniques to stabilize the RNA hairpins by linking the free ends of the RNA strand at the base of the hairpin stem. One method involves stilbene diether or hexaethylene glycol linkers and circularization by T4 RNA ligase. Another method uses click chemistry to stitch the RNA ends with a triazole linker. Both techniques are efficient and easy to perform. They should be useful in making stable, biologically relevant RNA constructs for structural studies.

Oxyonium phosphobetaines – unusually stable nucleophilic catalyst–phosphate complexes formed from H-phosphonates and N-oxides

Aryl H-phosphonates react with N-oxides to form previously unknown stable zwitterionic oxyonium phosphates comprising an −O–P–O–N+Z atom system. Their structures were confirmed i.e. by X-ray crystal structure analysis, and some mechanisms were proposed for their formation. Stability during storage and reactivity toward nucleophiles points to their possible synthetic applications.