Structural and Functional Aspects of G-Quadruplex Aptamers Which Bind a Broad Range of Influenza A Viruses

An aptamer is a synthetic oligonucleotide with a unique spatial structure that provides specific binding to a target. To date, several aptamers to hemagglutinin of the influenza A virus have been described, which vary in affinity and strain specificity. Among them, the DNA aptamer RHA0385 is able to recognize influenza hemagglutinins with highly variable sequences. In this paper, the structure of RHA0385 was studied by circular dichroism spectroscopy, nuclear magnetic resonance, and size-exclusion chromatography, demonstrating the formation of a parallel G-quadruplex structure. Three derivatives of RHA0385 were designed in order to determine the contribution of the major loop to affinity. Shortening of the major loop from seven to three nucleotides led to stabilization of the scaffold. The affinities of the derivatives were studied by surface plasmon resonance and an enzyme-linked aptamer assay on recombinant hemagglutinins and viral particles, respectively. The alterations in the loop affected the binding to influenza hemagglutinin, but did not abolish it. Contrary to aptamer RHA0385, two of the designed aptamers were shown to be conformationally homogeneous, retaining high affinities and broad binding abilities for both recombinant hemagglutinins and whole influenza A viruses.

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The Functional Role of Loops and Flanking Sequences of G-Quadruplex Aptamer to the Hemagglutinin of Influenza a Virus

International Journal of Molecular Sciences ◽

10.3390/ijms22052409 ◽

2021 ◽

Vol 22 (5) ◽

pp. 2409

Author(s):

Anastasia A. Bizyaeva ◽

Dmitry A. Bunin ◽

Valeria L. Moiseenko ◽

Alexandra S. Gambaryan ◽

Sonja Balk ◽

...

Keyword(s):

Influenza A Virus ◽

Influenza A ◽

Core Structure ◽

Dna Aptamer ◽

Tertiary Structures ◽

Quadruplex Dna ◽

G Quadruplex ◽

Flanking Sequences ◽

Related Proteins

Nucleic acid aptamers are generally accepted as promising elements for the specific and high-affinity binding of various biomolecules. It has been shown for a number of aptamers that the complexes with several related proteins may possess a similar affinity. An outstanding example is the G-quadruplex DNA aptamer RHA0385, which binds to the hemagglutinins of various influenza A virus strains. These hemagglutinins have homologous tertiary structures but moderate-to-low amino acid sequence identities. Here, the experiment was inverted, targeting the same protein using a set of related, parallel G-quadruplexes. The 5′- and 3′-flanking sequences of RHA0385 were truncated to yield parallel G-quadruplex with three propeller loops that were 7, 1, and 1 nucleotides in length. Next, a set of minimal, parallel G-quadruplexes with three single-nucleotide loops was tested. These G-quadruplexes were characterized both structurally and functionally. All parallel G-quadruplexes had affinities for both recombinant hemagglutinin and influenza virions. In summary, the parallel G-quadruplex represents a minimal core structure with functional activity that binds influenza A hemagglutinin. The flanking sequences and loops represent additional features that can be used to modulate the affinity. Thus, the RHA0385–hemagglutinin complex serves as an excellent example of the hypothesis of a core structure that is decorated with additional recognizing elements capable of improving the binding properties of the aptamer.

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Differential Behaviours and Preferential Bindings of Influenza Nucleoproteins on Importins-α

Viruses ◽

10.3390/v12080834 ◽

2020 ◽

Vol 12 (8) ◽

pp. 834 ◽

Cited By ~ 1

Author(s):

Amélie Donchet ◽

Emilie Vassal-Stermann ◽

Francine C. A. Gérard ◽

Rob W. H. Ruigrok ◽

Thibaut Crépin

Keyword(s):

Influenza A ◽

Influenza Viruses ◽

Size Exclusion ◽

High Association ◽

Intrinsically Disordered ◽

Specific Behaviour ◽

Exclusion Chromatography ◽

Translocation Mechanism ◽

Importin Α ◽

Localization Signal

Influenza viruses are negative single-stranded RNA viruses with nuclear transcription and replication. They enter the nucleus by using the cellular importin-α/-β nuclear import machinery. Influenza nucleoproteins from influenza A, B, C and D viruses possess a nuclear localization signal (NLS) localized on an intrinsically disordered extremity (NPTAIL). In this paper, using size exclusion chromatography (SEC), SEC-multi-angle laser light scattering (SEC-MALLS) analysis, surface plasmon resonance (SPR) and fluorescence anisotropy, we provide the first comparative study designed to dissect the interaction between the four NPTAILs and four importins-α identified as partners. All interactions between NPTAILs and importins-α have high association and dissociation rates and present a distinct and specific behaviour. D/NPTAIL interacts strongly with all importins-α while B/NPTAIL shows weak affinity for importins-α. A/NPTAIL and C/NPTAIL present preferential importin-α partners. Mutations in B/NPTAIL and D/NPTAIL show a loss of importin-α binding, confirming key NLS residues. Taken together, our results provide essential highlights of this complex translocation mechanism.

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Pandemic Avian Influenza and Intra/Interhaemagglutinin Subtype Electrostatic Variation among Viruses Isolated from Avian, Mammalian, and Human Hosts

BioMed Research International ◽

10.1155/2018/3870508 ◽

2018 ◽

Vol 2018 ◽

pp. 1-10 ◽

Cited By ~ 2

Author(s):

Irene Righetto ◽

Francesco Filippini

Keyword(s):

Avian Influenza ◽

Influenza A ◽

Specific Binding ◽

Antigenic Drift ◽

High Rate ◽

Influenza A Viruses ◽

Host Jump ◽

Viral Subtypes ◽

Host Jumping ◽

Novel Influenza A

Host jump can result in deadly pandemic events when avian influenza A viruses broaden their host specificity and become able to infect mammals, including humans. Haemagglutinin—the major capsid protein in influenza A viruses—is subjected to high rate mutations, of which several occur at its “head”: the receptor-binding domain that mediates specific binding to host cell receptors. Such surface-changing mutations may lead to antigenically novel influenza A viruses hence in pandemics by host jump and in vaccine escape by antigenic drift. Changes in haemagglutinin surface electrostatics have been recently associated with antigenic drift and with clades evolution and spreading in H5N1 and H9N2 viruses. We performed a comparative analysis of haemagglutinin surface electrostatics to investigate clustering and eventual fingerprints among representative pandemic (H5 and H7) and nonpandemic (H4 and H6) avian influenza viral subtypes. We observed preferential sorting of viruses isolated from mammalian/human hosts among these electrostatic clusters of a subtype; however, sorting was not “100% specific” to the different clusters. Therefore, electrostatic fingerprints can help in understanding, but they cannot explain alone the host jumping mechanism.

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Onchocerca retinol- and ivermectin-binding protein activity

Parasitology ◽

10.1017/s0031182000084791 ◽

1996 ◽

Vol 112 (2) ◽

pp. 221-225 ◽

Cited By ~ 8

Author(s):

P. G. Lal ◽

E. R. James

Keyword(s):

Adult Worm ◽

Specific Binding ◽

Binding Protein ◽

Binding Activity ◽

Retinol Binding Protein ◽

Size Exclusion ◽

Protein Activity ◽

Molecular Weights ◽

Molar Excess ◽

Exclusion Chromatography

SummaryThe presence of retinol-binding protein (RBP) activity in Onchocerca cervicalis adult worms and interaction with ivermectin has been studied using high pressure size exclusion chromatography (HPSEC). Four distinct peaks of [3H]-retinol incorporation were obtained corresponding to approximate molecular weights of 150, 67, 19·7 and 4–6 kDa, the 2 smaller Mr peaks accounting for most of the binding activity. Competition for binding using non-labelled retinol at 200-fold molar excess indicated that specific binding of retinol occurred only to the 19–7 kDa fraction. Competition by ivermectin also inhibited binding of [3H]-retinol to the third peak. Following incubation with [3H]-ivermectin & peaks of similar molecular weights were also detected by HPSEC in soluble adult worm homogenate, However, in this case the 150 kDa fraction was most prominent. Both non-labelled ivermectin and non-labelled retinol at 200-fold molar excess reduced binding of [3H]-ivermectin to all & fractions. These data indicate that the putative Onchocerca RBP has an approximate molecular weight of 19·7 kDa, that retinol also binds to 3 additional fractions non-specifically, that the pattern of binding of ivermectin to adult worm material is quantitatively and qualitatively different from the binding exhibited by retinol, and that ivermectin interferes with the binding of retinol to the 19·7 kDa Onchocerca protein.

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New G-Quadruplex-Forming Oligodeoxynucleotides Incorporating a Bifunctional Double-Ended Linker (DEL): Effects of DEL Size and ODNs Orientation on the Topology, Stability, and Molecularity of DEL-G-Quadruplexes

Molecules ◽

10.3390/molecules24030654 ◽

2019 ◽

Vol 24 (3) ◽

pp. 654 ◽

Cited By ~ 2

Author(s):

Maria Marzano ◽

Andrea Falanga ◽

Stefano D’Errico ◽

Brunella Pinto ◽

Giovanni Roviello ◽

...

Keyword(s):

Size Exclusion Chromatography ◽

Structural Characterization ◽

Size Exclusion ◽

Biological Processes ◽

Dna Nanostructures ◽

Nmr Studies ◽

Molecular Weights ◽

G Quadruplex ◽

Exclusion Chromatography

G-quadruplexes (G4s) are unusual secondary structures of DNA occurring in guanosine-rich oligodeoxynucleotide (ODN) strands that are extensively studied for their relevance to the biological processes in which they are involved. In this study, we report the synthesis of a new kind of G4-forming molecule named double-ended-linker ODN (DEL-ODN), in which two TG4T strands are attached to the two ends of symmetric, non-nucleotide linkers. Four DEL-ODNs differing for the incorporation of either a short or long linker and the directionality of the TG4T strands were synthesized, and their ability to form G4 structures and/or multimeric species was investigated by PAGE, HPLC–size-exclusion chromatography (HPLC–SEC), circular dichroism (CD), and NMR studies in comparison with the previously reported monomeric tetra-ended-linker (TEL) analogues and with the corresponding tetramolecular species (TG4T)4. The structural characterization of DEL-ODNs confirmed the formation of stable, bimolecular DEL-G4s for all DEL-ODNs, as well as of additional DEL-G4 multimers with higher molecular weights, thus suggesting a way towards the obtainment of thermally stable DNA nanostructures based on reticulated DEL-G4s.

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Variable-Temperature Size Exclusion Chromatography for the Study of the Structural Changes in G-Quadruplex

ISRN Biochemistry ◽

10.1155/2013/631875 ◽

2013 ◽

Vol 2013 ◽

pp. 1-7 ◽

Cited By ~ 1

Author(s):

Sanae Benabou ◽

Ramon Eritja ◽

Raimundo Gargallo

Keyword(s):

Size Exclusion Chromatography ◽

Promoter Region ◽

Structural Changes ◽

Variable Temperature ◽

Size Exclusion ◽

G Quadruplex ◽

Conformational Equilibria ◽

Exclusion Chromatography ◽

In The Wild ◽

Circular Dichroïsm

The conformational equilibria of a guanine-rich sequence found at the promoter region of the human c-kit oncogene are studied by means of circular dichroism spectroscopy (CD) and variable-temperature size exclusion chromatography (SEC). It is shown that the wild sequence ckit21 exists as a mixture of monomeric and multimeric G-quadruplexes. Appropriate mutation of several bases in the wild sequence produces the shift from parallel to antiparallel G-quadruplex, as well as the disappearance of multimeric species. The shift from the antiparallel to the parallel conformation induced by temperature is reflected in both CD and SEC profiles.

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A pH-dependent bolt involving cytosine bases located in the lateral loops of antiparallel G-quadruplex structures within the SMARCA4 gene promotor

Scientific Reports ◽

10.1038/s41598-019-52311-5 ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 3

Author(s):

Sanae Benabou ◽

Stefania Mazzini ◽

Anna Aviñó ◽

Ramon Eritja ◽

Raimundo Gargallo

Keyword(s):

Base Pair ◽

Thermodynamic Description ◽

Size Exclusion ◽

Spectroscopic Techniques ◽

G Quadruplex ◽

Exclusion Chromatography ◽

Additional Stability ◽

Gene Promotor ◽

Ph Dependent ◽

Melting Experiments

Abstract Some lung and ovarian tumors are connected to the loss of expression of SMARCA4 gene. In its promoter region, a 44-nucleotides long guanine sequence prone to form G-quadruplex structures has been studied by means of spectroscopic techniques (circular dichroism, molecular absorption and nuclear magnetic resonance), size exclusion chromatography and multivariate analysis. The results have shown that the central 21-nucleotides long sequence comprising four guanine tracts of disparate length is able to fold into a pH-dependent ensemble of G-quadruplex structures. Based on acid-base titrations and melting experiments of wild and mutated sequences, the formation of a C·C+ base pair between cytosine bases present at the two lateral loops is shown to promote a reduction in conformational heterogeneity, as well as an increase in thermal stability. The formation of this base pair is characterized by a pKa value of 7.1 ± 0.2 at 20 °C and 150 mM KCl. This value, higher than those usually found in i-motif structures, is related to the additional stability provided by guanine tetrads in the G-quadruplex. To our knowledge, this is the first thermodynamic description of this base pair in loops of antiparallel G-quadruplex structures.

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Cross-protective immunity of the hemagglutinin stalk domain presented on the surface of Lactococcus lactis against divergent influenza viruses in mice

10.21203/rs.3.rs-16035/v1 ◽

2020 ◽

Author(s):

Han Lei ◽

Tong Gao ◽

Qianhong Cen

Keyword(s):

Immune Responses ◽

Protective Immunity ◽

Influenza A ◽

Influenza Viruses ◽

Cross Protection ◽

Influenza A Viruses ◽

Universal Vaccine ◽

Influenza Hemagglutinin ◽

Alternative Approach ◽

Stalk Domain

Abstract Background Most of the current approaches to influenza vaccine design focus on antibodies against influenza hemagglutinin (HA). However, these influenza vaccines typically provide strain-specific protection against mostly homologous subtypes. There is an urgent need to develop a universal vaccine that confers cross-protection against influenza viruses. Of note, the HA stalk domain (HAs) is a new target for such an influenza vaccine.Results Recombinant L.lactis/pNZ8150-pgsA-HAs constructed in which pgsA was used as an anchor protein and investigated the immunogenicity of HAs, in the mouse model by oral administration without the use of a mucosal adjuvant. Mice were orally vaccinated with L.lactis/pNZ8150-pgsA-HAs, and then produced significant humoral and mucosal immune responses. Importantly, L.lactis/pNZ8150-pgsA-HAs provided significant cross-protection against H5N1, H3N2 or H1N1 virus infection.Conclusions Our data support the hypothesis that HAs presented on the surface of L. lactis can provide cross-protective immunity against influenza A viruses. Taken together, these findings suggest that L.lactis/pNZ8150-pgsA-HAs can be considered an alternative approach to developing a novel universal vaccine during an influenza A pandemic.

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N-glycolylneuraminic acid binding of avian H7 influenza A viruses

10.1101/2020.12.21.423767 ◽

2020 ◽

Author(s):

Cindy M. Spruit ◽

Xueyong Zhu ◽

Frederik Broszeit ◽

Alvin X. Han ◽

Roosmarijn van der Woude ◽

...

Keyword(s):

Amino Acids ◽

Cell Surface ◽

Influenza A Virus ◽

Influenza A ◽

Specific Binding ◽

Severe Illness ◽

Virus Species ◽

Influenza A Viruses ◽

Species Barrier ◽

Acetylneuraminic Acid

AbstractInfluenza A viruses initiate infection by binding to glycans with terminal sialic acids present on the cell surface. Hosts of influenza A viruses variably express two major forms of sialic acid, N-acetylneuraminic acid (NeuAc) and N-glycolylneuraminic acid (NeuGc). NeuGc is produced in the majority of mammals including horses, pigs, and mice, but is absent in humans, ferrets, and birds. Intriguingly, the only known naturally occurring influenza A viruses that exclusively bind NeuGc are the extinct highly pathogenic equine H7N7 viruses. We determined the crystal structure of a representative equine H7 hemagglutinin (HA) in complex with its NeuGc ligand and observed a high similarity in the receptor-binding domain with an avian H7 HA. To determine the molecular basis for NeuAc and NeuGc specificity, we performed systematic mutational analyses, based on the structural insights, on two distant avian H7 HAs. We found that mutation A135E is key for binding α2,3-linked NeuGc but does not abolish NeuAc binding. Interestingly, additional mutations S128T, I130V, or a combination of T189A and K193R, converted from NeuAc to NeuGc specificity as determined by glycan microarrays. However, specific binding to NeuGc-terminal glycans on our glycan array did not always correspond with full NeuGc specificity on chicken and equine erythrocytes and tracheal epithelium sections. Phylogenetic analysis of avian and equine H7 HAs that investigated the amino acids at positions 128, 130, 135, 189, and 193 reveals a clear distinction between equine and avian residues. The highest variability in amino acids (four different residues) is observed at key position 135, of which only the equine glutamic acid leads to binding of NeuGc. The results demonstrate that avian H7 viruses, although genetically distinct from equine H7 viruses, can bind NeuGc after the introduction of two to three mutations, providing insights into the adaptation of H7 viruses to NeuGc receptors.Author summaryInfluenza A viruses cause millions of cases of severe illness and deaths annually. To initiate infection and replicate, the virus first needs to bind to a structure on the cell surface, like a key fitting in a lock. For influenza A virus, these ‘keys’ (receptors) on the cell surface are chains of sugar molecules (glycans). The terminal sugar on these glycans is often either N-acetylneuraminic acid (NeuAc) or N-glycolylneuraminic acid (NeuGc). Most influenza A viruses bind NeuAc, but a small minority binds NeuGc. NeuGc is present in species like horses, pigs, and mice, but not in humans, ferrets, and birds. Therefore, NeuGc binding could be a determinant of an Influenza A virus species barrier. Here, we investigated the molecular determinants of NeuGc specificity and the origin of viruses that bind NeuGc.

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Influenza A M2 Channel Oligomerization is Sensitive to its Chemical Environment

10.1101/2021.05.07.443160 ◽

2021 ◽

Author(s):

Julia A Townsend ◽

Henry M Sanders ◽

Amber D Rolland ◽

James S Prell ◽

Jun Wang ◽

...

Keyword(s):

Mass Spectrometry ◽

Drug Targets ◽

Influenza A ◽

Matrix Protein ◽

Antiviral Drug ◽

Native Mass Spectrometry ◽

Chemical Environment ◽

Size Exclusion ◽

Oligomeric State ◽

Exclusion Chromatography

Viroporins are small viral ion channels that play important roles in the viral infection cycle and are proven antiviral drug targets. Matrix protein 2 from influenza A (AM2) is the best characterized viroporin, and the current paradigm is that AM2 forms monodisperse tetramers. Here, we used native mass spectrometry, ion mobility spectrometry, and size-exclusion chromatography to characterize the oligomeric state of full-length AM2 in a variety of different pH and detergent conditions. Unexpectedly, we discovered that AM2 formed a range of different oligomeric complexes that were strongly influenced by its local chemical environment. The monodisperse tetramer was only observed in select conditions when the antiviral drug, amantadine, was added. Native mass spectrometry of AM2 in lipid nanodiscs with different lipids showed that lipids also affected the oligomeric states of AM2. Finally, nanodiscs uniquely enabled measurement of amantadine binding stoichiometries to AM2 in the intact lipid bilayer. These unexpected results reveal that AM2 forms a wider range of oligomeric states than previously thought possible, which provides new potential mechanisms of influenza pathology and pharmacology.

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