Synthesis of a novel benzodifuran derivative and its molecular recognition of poly rA RNA

2013 ◽  
Vol 394 (9) ◽  
pp. 1235-1239 ◽  
Author(s):  
Giovanni N. Roviello ◽  
Valentina Roviello ◽  
Domenica Musumeci ◽  
Carlo Pedone
Keyword(s):  
Nucleic Acid ◽  
Chemical Synthesis ◽  
Conformational Changes ◽  
Cd Spectroscopy ◽  
Synthetic Approach ◽  
Stoichiometric Ratio ◽  
Dna And Rna ◽  
Nmr Characterization ◽  
Cd Studies ◽  
Heteroaromatic Molecule

Abstract In this manuscript, we describe a synthetic approach to a novel benzodifuran derivative as well as CD studies regarding its ability to interact with DNA and RNA. After the chemical synthesis and ESI-MS and NMR characterization, this heteroaromatic molecule was investigated by CD spectroscopy in order to evaluate it as a potential nucleic acid binder. Interestingly, the benzodifuran compound was found to be able to induce conformational changes in both DNA and RNA homoadenine molecules forming in the latter case a complex with a 6:1 benzodifuran/nucleobase stoichiometric ratio, as evidenced by CD titration experiments.

scholarly journals Structural Studies of HNA Substrate Specificity in Mutants of an Archaeal DNA Polymerase Obtained by Directed Evolution

Biomolecules ◽  
2020 ◽  
Vol 10 (12) ◽  
pp. 1647
Author(s):  
Camille Samson ◽  
Pierre Legrand ◽  
Mustafa Tekpinar ◽  
Jef Rozenski ◽  
Mikhail Abramov ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Dna Polymerase ◽  
Conformational Changes ◽  
Ternary Complex ◽  
Md Simulations ◽  
Binary And Ternary Complexes ◽  
Dna And Rna ◽  
Hybrid Duplex ◽  
Wide Range ◽  
New Generation

Archaeal DNA polymerases from the B-family (polB) have found essential applications in biotechnology. In addition, some of their variants can accept a wide range of modified nucleotides or xenobiotic nucleotides, such as 1,5-anhydrohexitol nucleic acid (HNA), which has the unique ability to selectively cross-pair with DNA and RNA. This capacity is essential to allow the transmission of information between different chemistries of nucleic acid molecules. Variants of the archaeal polymerase from Thermococcus gorgonarius, TgoT, that can either generate HNA from DNA (TgoT_6G12) or DNA from HNA (TgoT_RT521) have been previously identified. To understand how DNA and HNA are recognized and selected by these two laboratory-evolved polymerases, we report six X-ray structures of these variants, as well as an in silico model of a ternary complex with HNA. Structural comparisons of the apo form of TgoT_6G12 together with its binary and ternary complexes with a DNA duplex highlight an ensemble of interactions and conformational changes required to promote DNA or HNA synthesis. MD simulations of the ternary complex suggest that the HNA-DNA hybrid duplex remains stable in the A-DNA helical form and help explain the presence of mutations in regions that would normally not be in contact with the DNA if it were not in the A-helical form. One complex with two incorporated HNA nucleotides is surprisingly found in a one nucleotide-backtracked form, which is new for a DNA polymerase. This information can be used for engineering a new generation of more efficient HNA polymerase variants.

High-resolution nucleic acid sequence mapping via in situ hybridization at the Electron Microscope level

1995 ◽  
Vol 53 ◽  
pp. 752-753
Author(s):  
B.A. Hamkalo ◽  
S. Narayanswami ◽  
A.P. Kausch
Keyword(s):  
Nucleic Acid ◽  
Interphase Nucleus ◽  
Genome Mapping ◽  
Precise Location ◽  
Electron Microscope Level ◽  
Rna Sequences ◽  
Fundamental Interest ◽  
Whole Cells ◽  
Dna And Rna

The availability of nonradioactive methods to label nucleic acids an the resultant rapid and greater sensitivity of detection has catapulted the technique of in situ hybridization to become the method of choice to locate of specific DNA and RNA sequences on chromosomes and in whole cells in cytological preparations in many areas of biology. It is being applied to problems of fundamental interest to basic cell and molecular biologists such as the organization of the interphase nucleus in the context of putative functional domains; it is making major contributions to genome mapping efforts; and it is being applied to the analysis of clinical specimens. Although fluorescence detection of nucleic acid hybrids is routinely used, certain questions require greater resolution. For example, very closely linked sequences may not be separable using fluorescence; the precise location of sequences with respect to chromosome structures may be below the resolution of light microscopy(LM); and the relative positions of sequences on very small chromosomes may not be feasible.

scholarly journals Sulfur Modification in Natural RNA and Therapeutic Oligonucleotides

2021 ◽  
Author(s):  
Ya Ying Zheng ◽  
Ying Wu ◽  
Thomas Begley ◽  
Jia Sheng
Keyword(s):  
Nucleic Acid ◽  
Dna And Rna ◽  
Sulfur Modification ◽  
Oxygen Atoms

Sulfur modifications have been discovered on both DNA and RNA. Sulfur substitution of oxygen atoms at nucleobase or backbone locations in the nucleic acid framework led to a wide variety...

scholarly journals Recent developments in nucleic acid based techniques for use in rumen manipulation

2009 ◽  
Vol 38 (spe) ◽  
pp. 341-351 ◽  
Author(s):  
Christopher McSweeney ◽  
Seungha Kang ◽  
Emma Gagen ◽  
Carl Davis ◽  
Mark Morrison ◽  
...  
Keyword(s):  
Microbial Community ◽  
Nucleic Acid ◽  
Microbial Communities ◽  
Marker Gene ◽  
Molecular Ecology ◽  
Functional Gene ◽  
Rumen Microorganisms ◽  
Dna And Rna ◽  
Conventional Culture ◽  
Reference Strains

Nucleic acid-based techniques which can be used to characterise complex microbial communities without incubation are now being employed regularly in ruminant nutrition studies. Conventional culture-based methods for enumerating rumen microorganisms (bacteria, archaea, protozoa, and fungi) have been superseded and are now used mainly to obtain pure isolates of novel organisms and reference strains that are required for the development and validation of the nucleic acid approaches. These reference strains are also essential for physiological studies of the lifestyle of the organisms as well as sources of genomic DNA and RNA that can be analysed for functional gene activity. The foundation of the molecular ecology techniques is 16S/18S rDNA sequence analysis which has provided a phylogenetically based classification scheme for enumeration and identification of microbial community members. The use of this marker gene in assays involving the use of single nucleic acid probes or primer sets is rapidly evolving to high throughput approaches such as microarray analysis and new generation sequencing technologies. While these analyses are very informative for determining the composition of the microbial community and monitoring changes in population size, they can only infer function based on these observations. The focus of nucleic acid research is now shifting to the functional analysis of the ecosystem which involves the measurement of functional genes and their expression in the predominant or specific members of the rumen microbial community. Functional gene studies are less developed than 16S rDNA-based analysis of community structure. Also for gene expression studies there are inherent problems involved in extracting high quality RNA from digesta, and priming cDNA synthesis from bacterial mRNA. This paper reviews nucleic acid based molecular methods which have recently been developed for studying the structure and function of rumen microbial communities.

Characterisation of the conformational changes in platelet factor 4 induced by polyanions: towards in vitro prediction of antigenicity

2014 ◽  
Vol 112 (07) ◽  
pp. 53-64 ◽  
Author(s):  
Sven Brandt ◽  
Krystin Krauel ◽  
Kay E. Gottschalk ◽  
Thomas Renné ◽  
Christiane A. Helm ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Structural Changes ◽  
Cd Spectroscopy ◽  
Platelet Factor 4 ◽  
Drug Induced ◽  
Platelet Factor ◽  
Preclinical Drug Development ◽  
Varying Length ◽  
Endogenous Proteins

SummaryHeparin-induced thrombocytopenia (HIT) is the most frequent drug-induced immune reaction affecting blood cells. Its antigen is formed when the chemokine platelet factor 4 (PF4) complexes with polyanions. By assessing polyanions of varying length and degree of sulfation using immunoassay and circular dichroism (CD)-spectroscopy, we show that PF4 structural changes resulting in antiparallel β-sheet content >30% make PF4/polyanion complexes antigenic. Further, we found that polyphosphates (polyP-55) induce antigenic changes on PF4, whereas fondaparinux does not. We provide a model suggesting that conformational changes exposing antigens on PF4/polyanion complexes occur in the hairpin involving AA 32–38, which form together with C-terminal AA (66–70) of the adjacent PF4 monomer a continuous patch on the PF4 tetramer surface, explaining why only tetrameric PF4 molecules express “HIT antigens”. The correlation of antibody binding in immunoassays with PF4 structural changes provides the intriguing possibility that CD-spectroscopy could become the first antibody-independent, in vitro method to predict potential immunogenicity of drugs. CD-spectroscopy could identify compounds during preclinical drug development that induce PF4 structural changes correlated with antigenicity. The clinical relevance can then be specifically addressed during clinical trials. Whether these findings can be transferred to other endogenous proteins requires further studies.

scholarly journals Cleavage of DNA and RNA by PLD3 and PLD4 limits autoinflammatory triggering by multiple sensors

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Amanda L. Gavin ◽  
Deli Huang ◽  
Tanya R. Blane ◽  
Therese C. Thinnes ◽  
Yusuke Murakami ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Inflammatory Diseases ◽  
Type I ◽  
Multiple Sensors ◽  
Dna And Rna ◽  
Elevated Type ◽  
Minor Contribution ◽  
Ifn Γ ◽  
A Minor ◽  
Rna And Dna

AbstractPhospholipase D3 (PLD3) and PLD4 polymorphisms have been associated with several important inflammatory diseases. Here, we show that PLD3 and PLD4 digest ssRNA in addition to ssDNA as reported previously. Moreover, Pld3−/−Pld4−/− mice accumulate small ssRNAs and develop spontaneous fatal hemophagocytic lymphohistiocytosis (HLH) characterized by inflammatory liver damage and overproduction of Interferon (IFN)-γ. Pathology is rescued in Unc93b13d/3dPld3−/−Pld4−/− mice, which lack all endosomal TLR signaling; genetic codeficiency or antibody blockade of TLR9 or TLR7 ameliorates disease less effectively, suggesting that both RNA and DNA sensing by TLRs contributes to inflammation. IFN-γ made a minor contribution to pathology. Elevated type I IFN and some other remaining perturbations in Unc93b13d/3dPld3−/−Pld4−/− mice requires STING (Tmem173). Our results show that PLD3 and PLD4 regulate both endosomal TLR and cytoplasmic/STING nucleic acid sensing pathways and have implications for the treatment of nucleic acid-driven inflammatory disease.

scholarly journals Evidence that the TRPV1 S1-S4 Membrane Domain Contributes to Thermosensing

2019 ◽  
Author(s):  
Minjoo Kim ◽  
Nicholas J. Sisco ◽  
Jacob K. Hilton ◽  
Camila M. Montano ◽  
Manuel A. Castro ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Solution Nmr ◽  
Membrane Domain ◽  
Temperature Dependent ◽  
Nmr Characterization ◽  
Significant Interest ◽  
Pore Domain ◽  
Coupling Mechanisms

AbstractSensing and responding to temperature is crucial in biology. The TRPV1 ion channel is a well-studied heat-sensing receptor that is also activated by vanilloid compounds including capsaicin. Despite significant interest, the molecular underpinnings of thermosensing have remained elusive. The TRPV1 S1-S4 membrane domain couples chemical ligand binding to the pore domain during channel gating. However, the role of the S1-S4 domain in thermosensing is unclear. Evaluation of the isolated human TRPV1 S1-S4 domain by solution NMR, Far-UV CD, and intrinsic fluorescence shows that this domain undergoes a non-denaturing temperature-dependent transition with a high thermosensitivity. Further NMR characterization of the temperature-dependent conformational changes suggests the contribution of the S1-S4 domain to thermosensing shares features with known coupling mechanisms between this domain with ligand and pH activation. Taken together, this study shows that the TRPV1 S1-S4 domain contributes to TRPV1 temperature-dependent activation.

scholarly journals A Straightforward Methodology to Overcome Solubility Challenges for N-Terminal Cysteinyl Peptide Segments Used in Native Chemical Ligation

2020 ◽  
Author(s):  
Skander Abboud ◽  
El hadji Cisse ◽  
Michel Doudeau ◽  
Hélène Bénédetti ◽  
Vincent AUCAGNE
Keyword(s):  
Amino Acids ◽  
Chemical Synthesis ◽  
Building Blocks ◽  
Native Chemical Ligation ◽  
Synthetic Approach ◽  
Chemical Ligation ◽  
Limited Solubility ◽  
Peptide Segments

One of the main limitations encountered during the chemical synthesis of proteins through native chemical ligation (NCL) is the limited solubility of some of the peptide segments. The most commonly used solution to overcome this problem is to derivatize the segment with a temporary solubilizing tag. Conveniently, the tag can be introduced on the thioester segment in such a way that it is removed concomitantly with the NCL reaction. We herein describe a generalization of this approach to N-terminal cysteinyl segment counterparts, using a straightforward synthetic approach that can be easily automated from commercially available building blocks, and applied it to a well-known problematic target, SUMO-2 (93 amino acids).

scholarly journals Skipping and sliding to optimize target search on protein-bound DNA and RNA

2020 ◽  
Author(s):  
Misha Klein ◽  
Tao Ju Cui ◽  
Ian MacRae ◽  
Chirlmin Joo ◽  
Martin Depken
Keyword(s):  
Nucleic Acid ◽  
Single Molecule ◽  
Diffusion Limit ◽  
Optimal Search ◽  
Dna And Rna ◽  
Argonaute Proteins ◽  
Large Pool ◽  
Search Speed ◽  
Crowded Environments ◽  
Acid Substrate

Rapidly finding a specific nucleic-acid sequences in a large pool of competing off-targets is a fundamental challenge overcome by all living systems. To optimize the search and beat the diffusion limit, it is known that searchers should spend time sliding along the nucleic-acid substrate. Still, such sliding generally has to contend with high levels of molecular crowding on the substrate, and it remains unclear what effect this has on optimal search strategies. Using mechanistic modelling informed by single-molecule data, we show how sliding combined with correlated short-ranged skips allow searchers to maintain search speed on densely crowded substrates. We determine the conditions of optimal search, which show that an optimized searchers always spend more than half its time skipping and sliding along the substrate. Applying our theory to single-molecule data, we determine that both human and bacterial Argonaute proteins alternate between sliding 10 nt and skipping 30 nt along the substrate. We show that this combination of skipping and sliding lengths allows the searcher to maintain search speeds largely unaffected by molecular roadblocks covering up to 70% of the substrate. Our novel combination of experimental and theoretical approach could also help elucidate how other systems ensure rapid search in crowded environments.

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