NMR of RNA - Structure and interactions.

RNA was shown to have a more substantial role in the regulation of diverse cellular processes than anticipated until recently. Answers to questions what is the structure of specific RNAs, how structure changes to accommodate different functional roles, and how RNA senses other biomolecules and changes its fold upon interaction create a complete representation of RNA involved in cellular processes. Nuclear magnetic resonance (NMR) spectroscopy encompasses a collection of methods and approaches that offer insight into several structural aspects of RNAs. We review the most recent advances in the field of viral, long non-coding, regulatory, and four-stranded RNAs, with an emphasis on the detection of dynamic sub-states and in view of chemical modifications that expand RNA's function.

Enzymatic incorporation of an isotope-labeled adenine into RNA for the study of conformational dynamics by NMR.

Solution NMR spectroscopy is a well-established tool with unique advantages for structural studies of RNA molecules. However, for large RNA sequences, the NMR resonances often overlap severely. A reliable way to perform resonance assignment and allow further analysis despite spectral crowding is the use of site-specific isotope labeling in sample preparation. While solid-phase oligonucleotide synthesis has several advantages, RNA length and availability of isotope-labeled building blocks are persistent issues. Purely enzymatic methods represent an alternative and have been presented in the literature. In this study, we report on a method in which we exploit the preference of T7 RNA polymerase for nucleotide monophosphates over triphosphates for the 5' position, which allows 5'-labeling of RNA. Successive ligation to an unlabeled RNA strand generates a site-specifically labeled RNA. We show the successful production of such an RNA sample for NMR studies, report on experimental details and expected yields, and present the surprising finding of a previously hidden set of peaks which reveals conformational exchange in the RNA structure. This study highlights the feasibility of site-specific isotope-labeling of RNA with enzymatic methods.

Human Virus Genomes Are Enriched in Conserved Adenine/Thymine/Uracil Multiple Tracts That Pause Polymerase Progression.

The DNA secondary structures that deviate from the classic Watson and Crick base pairing are increasingly being reported to form transiently in the cell and regulate specific cellular mechanisms. Human viruses are cell parasites that have evolved mechanisms shared with the host cell to support their own replication and spreading. Contrary to human host cells, viruses display a diverse array of nucleic acid types, which include DNA or RNA in single-stranded or double-stranded conformations. This heterogeneity improves the possible occurrence of non-canonical nucleic acid structures. We have previously shown that human virus genomes are enriched in G-rich sequences that fold in four-stranded nucleic acid secondary structures, the G-quadruplexes.Here, by extensive bioinformatics analysis on all available genomes, we showed that human viruses are enriched in highly conserved multiple A (and T or U) tracts, with such an array that they could in principle form quadruplex structures. By circular dichroism, NMR, and Taq polymerase stop assays, we proved that, while A/T/U-quadruplexes do not form, these tracts still display biological significance, as they invariably trigger polymerase pausing within two bases from the A/T/U tract. "A" bases display the strongest effect. Most of the identified A-tracts are in the coding strand, both at the DNA and RNA levels, suggesting their possible relevance during viral translation. This study expands on the presence and mechanism of nucleic acid secondary structures in human viruses and provides a new direction for antiviral research.

Fused in Liposarcoma Protein, a New Player in the Regulation of HIV-1 Transcription, Binds to Known and Newly Identified LTR G-Quadruplexes.

HIV-1 integrated long terminal repeat (LTR) promoter activity is modulated by folding of its G-rich region into non-canonical nucleic acids structures, such as G-quadruplexes (G4s), and their interaction with cellular proteins. Here, by a combined pull-down/mass spectrometry/Western-blot approach, we identified the fused in liposarcoma (FUS) protein and found it to preferentially bind and stabilize the least stable and bulged LTR G4, especially in the cell environment. The outcome of this interaction is the down-regulation of viral transcription, as assessed in a reporter assay with LTR G4 mutants in FUS-silencing conditions. These data indicate that the complexity and dynamics of HIV-1 LTR G4s are much greater than previously envisaged. The G-rich LTR region, with its diverse G4 landscape and multiple cell protein interactions, stands out as prime sensing center for the fine regulation of viral transcription. This region thus represents a rational antiviral target for inhibiting both the actively transcribing and latent viruses.

The MDM2 inducible promoter folds into four-tetrad antiparallel G-quadruplexes targetable to fight malignant liposarcoma.

Well-differentiated liposarcoma (WDLPS) is a malignant neoplasia hard to diagnose and treat. Its main molecular signature is amplification of the MDM2-containing genomic region. The MDM2 oncogene is the master regulator of p53: its overexpression enhances p53 degradation and inhibits apoptosis, leading to the tumoral phenotype. Here, we show that the MDM2 inducible promoter G-rich region folds into stable G-quadruplexes both in vitro and in vivo and it is specifically recognized by cellular helicases. Cell treatment with G-quadruplex-ligands reduces MDM2 expression and p53 degradation, thus stimulating cancer cell cycle arrest and apoptosis. Structural characterization of the MDM2 G-quadruplex revealed an extraordinarily stable, unique four-tetrad antiparallel dynamic conformation, amenable to selective targeting. These data indicate the feasibility of an out-of-the-box G-quadruplex-targeting approach to defeat WDLPS and all tumours where restoration of wild-type p53 is sought. They also point to G-quadruplex-dependent genomic instability as possible cause of MDM2 expansion and WDLPS tumorigenesis.

Towards Understanding of Polymorphism of the G-rich Region of Human Papillomavirus Type 52.

The potential to affect gene expression via G-quadruplex stabilization has been extended to all domains of life, including viruses. Here, we investigate the polymorphism and structures of G-quadruplexes of the human papillomavirus type 52 with UV, CD and NMR spectroscopy and gel electrophoresis. We show that oligonucleotide with five G-tracts folds into several structures and that naturally occurring single nucleotide polymorphisms (SNPs) have profound effects on the structural polymorphism in the context of G-quadruplex forming propensity, conformational heterogeneity and folding stability. With help of SNP analysis, we were able to select one of the predominant forms, formed by G-rich sequence d(G3TAG3CAG4ACACAG3T). This oligonucleotide termed HPV52(1-4) adopts a three G-quartet snap back (3 + 1) type scaffold with four syn guanine residues, two edgewise loops spanning the same groove, a no-residue V loop and a propeller type loop. The first guanine residue is incorporated in the central G-quartet and all four-guanine residues from G4 stretch are included in the three quartet G-quadruplex core. Modification studies identified several structural elements that are important for stabilization of the described G-quadruplex fold. Our results expand set of G-rich targets in viral genomes and address the fundamental questions regarding folding of G-rich sequences.

RNA Dynamics by NMR Spectroscopy.

An ever-increasing number of functional RNAs require a mechanistic understanding. RNA function relies on changes in its structure, so-called dynamics. To reveal dynamic processes and higher energy structures, new NMR methods have been developed to elucidate these dynamics in RNA with atomic resolution. In this Review, we provide an introduction to dynamics novices and an overview of methods that access most dynamic timescales, from picoseconds to hours. Examples are provided as well as insight into theory, data acquisition and analysis for these different methods. Using this broad spectrum of methodology, unprecedented detail and invisible structures have been obtained and are reviewed here. RNA, though often more complicated and therefore neglected, also provides a great system to study structural changes, as these RNA structural changes are more easily defined-Lego like-than in proteins, hence the numerous revelations of RNA excited states.

A guide to large-scale RNA sample preparation.

RNA is becoming more important as an increasing number of functions, both regulatory and enzymatic, are being discovered on a daily basis. As the RNA boom has just begun, most techniques are still in development and changes occur frequently. To understand RNA functions, revealing the structure of RNA is of utmost importance, which requires sample preparation. We review the latest methods to produce and purify a variation of RNA molecules for different purposes with the main focus on structural biology and biophysics. We present a guide aimed at identifying the most suitable method for your RNA and your biological question and highlighting the advantages of different methods. Graphical abstract In this review we present different methods for large-scale production and purification of RNAs for structural and biophysical studies.

The effect of single nucleotide polymorphisms in G-rich regions of high-risk human papillomaviruses on structural diversity of DNA.

BACKGROUND: Infection with high-risk human papillomaviruses (HPVs) can lead to development of cancer of the head and neck and anogenital regions. G-rich sequences found in genomes of high-risk HPVs can fold into non-canonical secondary structures that could serve as 3D motifs distinct from double-stranded DNA and present recognition sites for ligands and opportunity for gene expression modulation. METHODS: Combination of UV, CD and NMR spectroscopy and PAGE electrophoresis were used as they offer complementary insights into structural changes of G-rich oligonucleotides. RESULTS: G-rich region of HPV16 is shown to preferentially form hairpin structures, while regions of HPV18, HPV52 and HPV58 fold into four-stranded DNA structures called G-quadruplexes. Single nucleotide polymorphisms found in G-rich sequences have been found to promote formation of hairpin structures of HPV16 and have affected number of species formed in G-rich region of HPV52, whereas they have exhibited minimal effect on the formation of HPV18 and HPV58 G-quadruplex structures. These structural changes were reflected in differences in apparent thermal stabilities. CONCLUSIONS: Potential of G-rich sequences as drug targets was evaluated based on the results of the current study. HPV16 and HPV18 are considered less appropriate targets due to several single nucleotide polymorphisms and low stability, respectively. On the other hand, HPV52 and HPV58 could be used for small-molecule mediated stabilization. GENERAL SIGNIFICANCE: G-rich sequences occurring in high-risk HPVs can fold into hairpin and G-quadruplex structures that could be potentially utilized as drug targets. This article is part of a Special Issue entitled "G-quadruplex" Guest Editor: Dr. Concetta Giancola and Dr. Daniela Montesarchio.

The Effect of DNA Sequence Directionality on G-Quadruplex Folding.

Sequence inversion in G-rich DNA from 5'→3' to 3'→5' exerts a substantial effect on the number of structures formed, while the type of G-quadruplex fold is in fact determined by the presence of K(+) or Na(+) ions. The melting temperatures of G-quadruplexes adopted by oligonucleotides with sequences in the 5'→3' direction are higher than those of their 3'→5' counterparts with both KCl and NaCl. CD, UV, and NMR spectroscopy demonstrates the importance of primary sequence for the structural diversity of G-quadruplexes. The changes introduced by mere sequence reversal of the G-rich DNA segment have a substantial impact on the polymorphic nature of the resulting G-quadruplexes and their potential physiological roles. The insights resulting from this study should enable extension of the empirical rules for the prediction of G-quadruplex topology.

Listeriolysin O Affects the Permeability of Caco-2 Monolayer in a Pore-Dependent and Ca2+-Independent Manner.

Listeria monocytogenes is a food and soil-borne pathogen that secretes a pore-forming toxin listeriolysin O (LLO) as its major virulence factor. We tested the effects of LLO on an intestinal epithelial cell line Caco-2 and compared them to an unrelated pore-forming toxin equinatoxin II (EqtII). Results showed that apical application of both toxins causes a significant drop in transepithelial electrical resistance (TEER), with higher LLO concentrations or prolonged exposure time needed to achieve the same magnitude of response than with EqtII. The drop in TEER was due to pore formation and coincided with rearrangement of claudin-1 within tight junctions and associated actin cytoskeleton; however, no significant increase in permeability to fluorescein or 3 kDa FITC-dextran was observed. Influx of calcium after pore formation affected the magnitude of the drop in TEER. Both toxins exhibit similar effects on epithelium morphology and physiology. Importantly, LLO action upon the membrane is much slower and results in compromised epithelium on a longer time scale at lower concentrations than EqtII. This could favor listerial invasion in hosts resistant to E-cadherin related infection.

Targeting VEGF with LNA-stabilized G-rich oligonucleotide for efficient breast cancer inhibition.

In this study, we investigated the efficacy of an LNA (locked nucleic acid)-modified DNA aptamer named RNV66 targeting VEGF against various breast cancer cell lines. Our results demonstrate that RNV66 efficiently inhibits breast cancer cell proliferation both in vitro and in vivo. Introduction of LNA nucleotides were crucial for higher efficacy. Furthermore, the binding interaction of RNV66 with VEGF was investigated using molecular dynamic simulations leading to the first computational model of the LNA aptamer-VEGF complex blocking its interaction with VEGF-receptor.

Structural foundation for DNA behavior in hydrated ionic liquid: An NMR study.

A well known rule of high thermal stability of GC-rich DNA helices can be reversed with the use of certain ions, rendering AT-rich duplexes more stable. We have sought to elucidate the structural basis of this phenomenon for choline dihydrogen phosphate, an ionic liquid known for extension of long-term chemical stability of biomolecules. NMR experiments complemented with CD spectroscopy revealed subtle changes of GC and AT-rich double helix structures in choline dihydrogen phosphate compared to NaCl solution. Chemical shift changes observed for different environments were used as a guide to determine choline ions' localization hotspots. For d(5'-AAATATATTT-3') choline ions are localized in the central part, especially in the minor groove near sugar protons of thymidine and H2 protons of adenine residues. In agreement with NMR data, thermodynamic analysis points to the involvement of choline ions in the hydration network as a crucial part of thermal stabilization of AT-rich helices. Analysis for GC-rich d(5'-GGGCGCGCCC-3') oligonucleotide showed preference of choline ions for major groove with less clearly defined localizations spots than in the case of its AT-rich counterpart.

New assay for quantification of PEGylated proteins during in vitro permeability studies.

One of the major challenges when analyzing very low amounts of PEGylated proteins is finding a sensitive analytical method. Immunoassays are most frequently used, however, conjugation can partially or completely mask protein epitopes, which can substantially lower the response and influence the quantitation range. Here we describe a novel assay that allows quantification of low amounts of PEGylated or differently conjugated proteins. The basic principle is similar to the classic sandwich ELISA but there are no antibodies used neither for capture nor for detection. Instead, Ni(2+) chelation is exploited for capture and affinity between streptavidin and biotin for the detection step. The usefulness of the assay was proven in permeation studies (Caco-2 cell model) using diversely conjugated TNF-a protein. This approach could be extended to numerous other proteins eliminating the need to develop a separate assay for each protein/project.

Calu-3 model under AIC and LCC conditions and application for protein permeability studies.

Broad area of respiratory epithelium with mild surface conditions is an attractive possibility when trans-mucosal delivery of protein drugs is considered. A mucus and cellular barrier of respiratory epithelium can be modelled in vitro by Calu-3 cell line. We have monitored morphology and barrier properties of Calu-3 culture on permeable supports while developing into liquid covered or air interfaced and mucus lined cellular barrier. Besides morphological differences, cultures differed in electrical resistance and permeability to proteins as well. The accelerated permeability to proteins in these models, due to permeability modulator MP C16, was examined. The effect on electrical resistance of cellular layer was rapid in both cultures suggesting easy access of MP C16 to cells even though its overall impact on cell permeability was strongly reduced in mucus covered culture. Differences in properties of the two models enable better understanding of protein transmucosal permeability, suggesting route of transport and MP C16 modulator action.

Human papillomavirus G-quadruplexes.

Infection with human papillomaviruses (HPVs) is one of the most common sexually transmitted infections and can lead to development of head and neck, skin, and anogenital cancer, including cervical cancer, which represents one of the world's most significant health problems. In this study, we analyze G-rich regions in all known HPV genomes in order to evaluate their potential to fold into G-quadruplex structure. Interestingly, G-rich loci fulfilling the criteria for G-quadruplex formation were found in only 8 types of HPV. Nevertheless, viral G-quadruplexes in 7 sequences derived directly from HPVs are confirmed here for the first time. G-rich regions with the capacity to form G-quadruplexes are located in the LCR, L2, E1, and E4 regions of the HPV genome; therefore we assume that regulation processes in viruses could be affected by G-quadruplex formation. Our results represent a starting point for the design of specific ligands with viral G-quadruplex motifs and suggest novel methods for the control of viral replication and transcription.

G-rich VEGF aptamer with locked and unlocked nucleic acid modifications exhibits a unique G-quadruplex fold.

The formation of a single G-quadruplex structure adopted by a promising 25 nt G-rich vascular endothelial growth factor aptamer in a K(+) rich environment was facilitated by locked nucleic acid modifications. An unprecedented all parallel-stranded monomeric G-quadruplex with three G-quartet planes exhibits several unique structural features. Five consecutive guanine residues are all involved in G-quartet formation and occupy positions in adjacent DNA strands, which are bridged with a no-residue propeller-type loop. A two-residue D-shaped loop facilitates inclusion of an isolated guanine residue into the vacant spot within the G-quartet. The remaining two G-rich tracts of three residues each adopt parallel orientation and are linked with edgewise and propeller loops. Both 5' with 3 nt and 3' with 4 nt overhangs display well-defined conformations, with latter adopting a basket handle topology. Locked residues contribute to thermal stabilization of the adopted structure and formation of structurally pre-organized intermediates that facilitate folding into a single G-quadruplex. Understanding the impact of chemical modifications on folding, thermal stability and structural polymorphism of G-quadruplexes provides means for the improvement of vascular endothelial growth factor aptamers and advances our insights into driving nucleic acid structure by locking or unlocking the conformation of sugar moieties of nucleotides in general.

The Caco-2 cell culture model enables sensitive detection of enhanced protein permeability in the presence of N-decyl-ß-d-maltopyranoside.

Appropriate assessment of transepithelial permeability in vitro is needed to estimate and model trans-mucosal bioavailability to achieve oral delivery of protein biopharmaceuticals. The Caco-2 cell-based intestinal epithelium model is widely used for this purpose for low molecular mass drugs. The aim of this study was to test the suitability of the Caco-2 model for assessing enhanced transepithelial permeability to proteins. Four unrelated proteins were chosen to test the permeability of Caco-2 monolayers. It was found that proteins could cross the epithelium model, in spite of their size. All tested proteins had similar very low apparent permeability coefficients (Papp) of around 4×10(-10)cm/s. Protein stability over three-hour exposure to Caco-2 was also confirmed. Their crossing rate in a cell-free setup was also measured, to determine the upper limit of permeability to proteins. An epithelium permeability enhancer N-decyl-ß-d-maltopyranoside (MP C10) was used to demonstrate accelerated permeability conditions. Papp values could be increased dose dependently up to about 1×10(-7)cm/s, close to the level in the cell-free setup, indicating distinctive potential of the model. This along with enhancing effect of known specific route permeators suggests involvement of the paracellular route in protein transport. Our results thus indicate that the Caco-2 model is a suitable tool for in vitro assessment of enhanced permeability to proteins.

Computational and experimental investigation of DNA repair protein photolyase interactions with low molecular weight drugs.

This paper reports the previously unknown interactions between eight low molecular weight commercially available drugs (130-800 Da) and DNA repair protein photolyase using computational docking simulations and surface plasmon resonance (SPR) experiments. Theoretical dissociation constants, K(d), obtained from molecular docking simulations were compared with the values found from SPR experiments. Among the eight drugs analyzed, computational and experimental values showed similar binding affinities between selected drug and protein pairs. We found no significant differences in binding interactions between pure and commercial forms of the drug lornoxicam and DNA photolyase. Among the eight drugs studied, prednisone, desloratadine, and azelastine exhibited the highest binding affinity (K(d) = 1.65, 2.05, and 8.47 µM, respectively) toward DNA photolyase. Results obtained in this study are promising for use in the prediction of unknown interactions of common drugs with specific proteins such as human clock protein cryptochrome.

Solution-state structure of an intramolecular G-quadruplex with propeller, diagonal and edgewise loops.

We herein report on the formation and high-resolution NMR solution-state structure determination of a G-quadruplex adopted by d[G(3)ATG(3)ACACAG(4)ACG(3)] comprised of four G-tracts with the third one consisting of four guanines that are intervened with non-G streches of different lengths. A single intramolecular antiparallel (3+1) G-quadruplex exhibits three stacked G-quartets connected with propeller, diagonal and edgewise loops of different lengths. The propeller and edgewise loops are well structured, whereas the longer diagonal loop is more flexible. To the best of our knowledge, this is the first high-resolution G-quadruplex structure where all of the three main loop types are present.