An integrated perspective on RNA aptamer ligand-recognition models : clearing muddy waters

Riboswitches are short RNA motifs that sensitively and selectively bind cognate ligands to modulate gene expression. Like protein receptor-ligand pairs, their binding dynamics are traditionally categorized as following one of two paradigmatic mechanisms: conformational selection and induced fit. In conformational selection, ligand binding stabilizes a particular state already present in the receptor’s dynamic ensemble. In induced fit, ligand-receptor interactions enable the system to overcome the energetic barrier into a previously inaccessible state. In this article, we question whether a polarized division of RNA binding mechanisms truly meets the conceptual needs of the field. We will review the history behind this classification of RNA-ligand interactions, and the way induced fit in particular has been rehabilitated by single-molecule studies of RNA aptamers. We will highlight several recent results from single-molecule experimental studies of riboswitches that reveal gaps or even contradictions between common definitions of the two terms, and we will conclude by proposing a more robust framework that considers the range of RNA behaviors unveiled in recent years as a reality to be described, rather than an increasingly unwieldy set of exceptions to the traditional models.PostprintPeer reviewe

Binding dynamics of a monomeric SSB protein to DNA : a single-molecule multi-process approach

People Programme of the European Union’s Seventh Framework Programme [REA 334496 to B.E.B.]; Leonardo da Vinci European Union Programme (to M.F.G.); Wellcome Trust [099149/Z/12/Z, 091825/Z/10/Z]. Funding for open access charge: Wellcome Trust; University of St Andrews.Single-stranded DNA binding proteins (SSBs) are ubiquitous across all organisms and are characterized by the presence of an OB (oligonucleotide/oligosaccharide/oligopeptide) binding motif to recognize single-stranded DNA (ssDNA). Despite their critical role in genome maintenance, our knowledge about SSB function is limited to proteins containing multiple OB-domains and little is known about single OB-folds interacting with ssDNA. Sulfolobus solfataricus SSB (SsoSSB) contains a single OB-fold and being the simplest representative of the SSB-family may serve as a model to understand fundamental aspects of SSB:DNA interactions. Here, we introduce a novel approach based on the competition between Förster resonance energy transfer (FRET), protein-induced fluorescence enhancement (PIFE) and quenching to dissect SsoSSB binding dynamics at single monomer resolution. We demonstrate that SsoSSB follows a monomer-by-monomer binding mechanism that involves a positive-cooperativity component between adjacent monomers. We found that SsoSSB dynamic behaviour is closer to that of Replication Protein A than to Escherichia coli SSB; a feature that might be inherited from the structural analogies of their DNA-binding domains. We hypothesize that SsoSSB has developed a balance between highdensity binding and a highly dynamic interaction with ssDNA to ensure efficient protection of the genome but still allow access to ssDNA during vital cellular processes.Publisher PDFPeer reviewe

Fluorescence-based strategies to investigate the structure and dynamics of aptamer-ligand complexes

This work was funded by the Scottish Universities Physics Alliance (SUPA), the University of St Andrews, the Engineering and Physical Sciences ResearchCouncil (EPSRC), the Canadian Institutes of Health Research (CIHR). Funding for open access charge: University of St Andrews.In addition to the helical nature of double-stranded DNA and RNA, single-stranded oligonucleotides can arrange themselves into tridimensional structures containing loops, bulges, internal hairpins and many other motifs. This ability has been used for more than two decades to generate oligonucleotide sequences, so-called aptamers, that can recognize certain metabolites with high affinity and specificity. More recently, this library of artificially-generated nucleic acid aptamers has been expanded by the discovery that naturally occurring RNA sequences control bacterial gene expression in response to cellular concentration of a given metabolite. The application of fluorescence methods has been pivotal to characterize in detail the structure and dynamics of these aptamer-ligand complexes in solution. This is mostly due to the intrinsic high sensitivity of fluorescence methods and also to significant improvements in solid-phase synthesis, post-synthetic labelling strategies and optical instrumentation that took place during the last decade. In this work, we provide an overview of the most widely employed fluorescence methods to investigate aptamer structure and function by describing the use of aptamers labelled with a single dye in fluorescence quenching and anisotropy assays. The use of 2-aminopurine as a fluorescent analog of adenine to monitor local changes in structure and fluorescence resonance energy transfer (FRET) to follow long-range conformational changes is also covered in detail. The last part of the review is dedicated to the application of fluorescence techniques based on single-molecule microscopy, a technique that has revolutionized our understanding of nucleic acid structure and dynamics. We finally describe the advantages of monitoring ligand-binding and conformational changes, one molecule at a time, to decipher the complexity of regulatory aptamers and summarize the emerging folding and ligand-binding models arising from the application of these single-molecule FRET microscopy techniques.Publisher PDFPeer reviewe

O jogo da informação

TCC (graduação) - Universidade Federal de Santa Catarina. Centro de Comunicação e Expressão. Jornalismo.Usados de forma experimental e pouco inspirados em produzir informações jornalísticas, a maioria dos newsgames ainda são produtos muito novos no jornalismo mundial. Esta monografia, trabalho de conclusão de curso, estuda o newsgame 7 Ways to Defy Health, do jornal Washington Post, com o objetivo de identificar diferenças e semelhanças na forma como estruturam-se os fatos verídicos e os elementos de verossimilhança neste tipo de jogo considerando "jogos sérios". Para dar conta desta discussão, a teoria de base é a Semiótica Discursiva, articulada com a Teoria do Jornalismo, principalmente aqueles autores ligados aos estudos de newsgames, como Ian Bogost. Este trabalho de conclusão de curso quer contribuir para propor indicativos que auxiliem na produção de newsgames mais conectados com os objetivos do jornalismo contemporâneo

Association of acculturation with cardiac structure and function among Hispanics/Latinos: a cross-sectional analysis of the echocardiographic study of Latinos.

OBJECTIVE:Hispanics/Latinos, the largest immigrant population in the USA, undergo the process of acculturation and have a large burden of heart failure risk. Few studies have examined the association of acculturation on cardiac structure and function. DESIGN:Cross-sectional. SETTING:The Echocardiographic Study of Latinos. PARTICIPANTS:1818 Hispanic adult participants with baseline echocardiographic assessment and acculturation measured by the Short Acculturation Scale, nativity, age at immigration, length of US residence, generational status and language. PRIMARY AND SECONDARY OUTCOME MEASURES:Echocardiographic assessment of left atrial volume index (LAVI), left ventricular mass index (LVMI), early diastolic transmitral inflow and mitral annular velocities. RESULTS:The study population was predominantly Spanish-speaking and foreign-born with mean residence in the US of 22.7 years, mean age of 56.4 years; 50% had hypertension, 28% had diabetes and 44% had a body mass index &gt;30 kg/m2. Multivariable analyses demonstrated higher LAVI with increasing years of US residence. Foreign-born and first-generation participants had higher E/e' but lower LAVI and e' velocities compared with the second generation. Higher acculturation and income &gt;20K were associated with higher LVMI, LAVI and E/e' but lower e' velocities. Preferential Spanish-speakers with an income <20K had a higher E/e'. CONCLUSIONS:Acculturation was associated with abnormal cardiac structure and function, with some effect modification by socioeconomic status

Optimització d'una aplicacio bioinformàtica d'alineament de seqüències executada en processadors multi-core i many-core (GPUs)

Las aplicaciones de alineamiento de secuencias son una herramienta importante para la comunidad científica. Estas aplicaciones bioinformáticas son usadas en muchos campos distintos como pueden ser la medicina, la biología, la farmacología, la genética, etc. A día de hoy los algoritmos de alineamiento de secuencias tienen una complejidad elevada y cada día tienen que manejar un volumen de datos más grande. Por esta razón se deben buscar alternativas para que estas aplicaciones sean capaces de manejar el aumento de tamaño que los bancos de secuencias están sufriendo día a día. En este proyecto se estudian y se investigan mejoras en este tipo de aplicaciones como puede ser el uso de sistemas paralelos que pueden mejorar el rendimiento notablemente.Les aplicacions d'alineament de seqüències són una eina important per a la comunitat científica. Aquestes aplicacions bioinformàtiques són utilitzades en molts camps diferents com poden ser la medicina, la biologia, la farmacologia, la genètica, etc. A dia d'avui els algorismes d'alineament de seqüències tenen una complexitat elevada i cada dia han de gestionar un volum de dades més gran. Per això s'han de buscar alternatives per a que aquestes aplicacions siguin capaces de gestionar l'augment de mida que els bancs de seqüències estan patint dia a dia. En aquest projecte s'estudien i s'investiguen millores en aquest tipus d'aplicacions com pot ser l'ús de sistemes paral·leles que poden millorar el rendiment notablement.The sequence alignment applications are an important tool for the scientific community. These bioinformatics applications are used in many different fields such as medicine, biology, pharmacology, genetics, etc. Today the sequence alignment algorithms are highly complex and every day have to handle a large volume of data. For this reason we must find alternatives for these applications are able to handle the increased size of sequences that banks are suffering every day. In this project we study and investigate improvements in these applications such as the use of parallel systems that can improve performance significantly

Licitações, contratos e Modelo Brasileiro de Processo: notas sobre a viabilidade da utilização de ferramentas processuais para conferir maior eficiência às aquisições públicas

Este artigo tem por objetivo demonstrar que o rigor procedimental a que em geral se atribui a ineficiência das licitações/contratações públicas pode ser flexibilizado, de modo a produzir eficiência, mediante utilização de soluções típicas do Direito Processual. Analisamos os textos normativos disponíveis e fizemos cuidadosa revisão da literatura jurídica (fontes). Depois disso, conjugamos a doutrina da processualidade no Direito Administrativo à compreensão (inerente a ela e à doutrina do formalismo-valorativo) de que o processo se diferencia do procedimento por se qualificar como procedimento em contraditório (método), demonstrando que as licitações/contratações públicas se apresentam, sob a égide da Lei nº 14.133/2021, como autênticos processos administrativos, e que essa especificidade do seu regime normativo abre campo para a utilização de ferramentas próprias do Modelo Brasileiro de Processo para procurar superar a referida rigidez procedimental (resultados). A pesquisa não comporta limitações de ordem prática (porque todas as fontes consultadas estavam disponíveis) ou teórica (porque dialoga com publicações anteriores) relevantes. Além disso, ela tem implicações sociais importantes. Afinal, se é verdade que o rigor procedimental incidente nesse contexto pode comprometer a eficiência das aquisições públicas, também é certo que a atividade administrativa (inclusive aquela realizada nas licitações e contratos da Administração) não pode prescindir de um mínimo de rigor procedimental, sem o qual seria inviável o exercício da atividade de controle administrativo que possibilita à a prevenção de atos de corrupção e da malversação de recursos públicos. Disso resulta o valor e a originalidade da sua contribuição para o desenvolvimento da Ciência

Mechanism of DNA loading by the DNA repair helicase XPD

Funding: Welcome Trust Programme Grant [WT091825MA to M.F.W., J.H.N.]; Wellcome Trust [099149/Z/12/Z]; Royal Society Wolfson Merit Award (to M.F.W., J.H.N.). Funding for open access charge: Wellcome Trust [WT091825MA].The xeroderma pigmentosum group D (XPD) helicase is a component of the transcription factor IIH complex in eukaryotes and plays an essential role in DNA repair in the nucleotide excision repair pathway. XPD is a 5′ to 3′ helicase with an essential iron–sulfur cluster. Structural and biochemical studies of the monomeric archaeal XPD homologues have aided a mechanistic understanding of this important class of helicase, but several important questions remain open. In particular, the mechanism for DNA loading, which is assumed to require large protein conformational change, is not fully understood. Here, DNA binding by the archaeal XPD helicase from Thermoplasma acidophilum has been investigated using a combination of crystallography, cross-linking, modified substrates and biochemical assays. The data are consistent with an initial tight binding of ssDNA to helicase domain 2, followed by transient opening of the interface between the Arch and 4FeS domains, allowing access to a second binding site on helicase domain 1 that directs DNA through the pore. A crystal structure of XPD from Sulfolobus acidocaldiarius that lacks helicase domain 2 has an otherwise unperturbed structure, emphasizing the stability of the interface between the Arch and 4FeS domains in XPD.Publisher PDFPeer reviewe

Asymmetric base-pair opening drives helicase unwinding dynamics

The opening of a Watson-Crick double helix is required for crucial cellular processes, including replication, repair, and transcription. It has long been assumed that RNA or DNA base pairs are broken by the concerted symmetric movement of complementary nucleobases. By analyzing thousands of base-pair opening and closing events from molecular simulations, here, we uncover a systematic stepwise process driven by the asymmetric flipping-out probability of paired nucleobases. We demonstrate experimentally that such asymmetry strongly biases the unwinding efficiency of DNA helicases toward substrates that bear highly dynamic nucleobases, such as pyrimidines, on the displaced strand. Duplex substrates with identical thermodynamic stability are thus shown to be more easily unwound from one side than the other, in a quantifiable and predictable manner. Our results indicate a possible layer of gene regulation coded in the direction-dependent unwindability of the double helix

Conformational Rearrangements Regulating the DNA Repair Protein APE1

Apurinic apyrimidinic endonuclease 1 (APE1) is a key enzyme of the Base Excision Repair (BER) pathway, which primarily manages oxidative lesions of DNA. Once the damaged base is removed, APE1 recognises the resulting abasic site and cleaves the phosphodiester backbone to allow for the correction by subsequent enzymes of the BER machinery. In spite of a wealth of information on APE1 structure and activity, its regulation mechanism still remains to be understood. Human APE1 consists of a globular catalytic domain preceded by a flexible N-terminal extension, which might be involved in the interaction with DNA. Moreover, the binding of the nuclear chaperone nucleophosmin (NPM1) to this region has been reported to impact APE1 catalysis. To evaluate intra- and inter-molecular conformational rearrangements upon DNA binding, incision, and interaction with NPM1, we used F&ouml;rster resonance energy transfer (FRET), a fluorescence spectroscopy technique sensitive to molecular distances. Our results suggest that the N-terminus approaches the DNA at the downstream side of the abasic site and enables the building of a predictive model of the full-length APE1/DNA complex. Furthermore, the spatial configuration of the N-terminal tail is sensitive to NPM1, which could be related to the regulation of APE1.This research was funded by the University of the Basque Country (grant number GIU18/172). The APC was funded by the Basque Government (grant number IT1454-22)