Screening and Initial Binding Assessment of Fumonisin B1 Aptamers

Fumonisins are mycotoxins produced by Fusarium verticillioides and F. proliferatum, fungi that are ubiquitous in corn (maize). Insect damage and some other environmental conditions result in the accumulation of fumonisins in corn-based products worldwide. Current methods of fumonisin detection rely on the use of immunoaffinity columns and high-performance liquid chromatography (HPLC). The use of aptamers offers a good alternative to the use of antibodies in fumonisin cleanup and detection due to lower costs and improved stability. Aptamers are single-stranded oligonucleotides that are selected using Systematic Evolution of Ligands by EXponential enrichment (SELEX) for their ability to bind to targets with high affinity and specificity. Sequences obtained after 18 rounds of SELEX were screened for their ability to bind to fumonisin B1. Six unique sequences were obtained, each showing improved binding to fumonisin B1 compared to controls. Sequence FB1 39 binds to fumonisin with a dissociation constant of 100 ± 30 nM and shows potential for use in fumonisin biosensors and solid phase extraction columns

Covalent binding of the natural antimicrobial peptide indolicidin to DNA abasic sites

Indolicidin is a host defense tridecapeptide that inhibits the catalytic activity of HIV-1 integrase in vitro. Here we have elucidated its mechanism of integrase inhibition. Using crosslinking and mass spectrometric footprinting approaches, we found that indolicidin interferes with formation of the catalytic integrase-DNA complex by directly binding DNA. Further characterization revealed that the peptide forms covalent links with abasic sites. Indolicidin crosslinks single- or double-stranded DNAs and various positions of the viral cDNA with comparable efficiency. Using truncated and chemically modified peptides, we show that abasic site crosslinking is independent of the PWWP motif but involves the indolicidin unique lysine residue and the N- and C- terminal NH(2) groups. Because indolicidin can also inhibit topoisomerase I, we believe that multiple actions at the level of DNA might be a common property of antimicrobial peptides

Novel bimodular DNA aptamers with guanosine quadruplexes inhibit phylogenetically diverse HIV-1 reverse transcriptases

DNA aptamers RT5, RT6 and RT47 form a group of related sequences that inhibit HIV-1 reverse transcriptase (RT). The essential inhibitory structure is identified here as bimodular, with a 5′ stem–loop module physically connected to a 3′-guanosine quadruplex module. The stem–loop tolerates considerable sequence plasticity. Connections between the guanosine triplets in the quadruplex could be simplified to a single nucleotide or a nonnucleic acid linker, such as hexaethylene glycol. All 12 quadruplex guanosines are required in an aptamer retaining most of the original loop sequence from RT6; only 11 are required for aptamer R1T (single T residue in intra-quadruplex loops). Circular dichroism (CD) spectroscopy gave ellipticity minima and maxima at 240 nm and 264 nm, indicating a parallel arrangement of the quadruplex strands. The simplified aptamers displayed increased overall stability. An aptamer carrying the original intra-quadruplex loops from RT6 inhibited RT in K+ buffers but not in Na+ buffers and displayed significant CD spectral broadening in Na+ buffers, while R1T inhibited RT in both buffers and displayed less broadening in Na+ buffers. The bimodular ssDNA aptamers inhibited RT from diverse primate lentiviruses with low nM IC50 values. These data provide insight into the requirements for broad-spectrum RT inhibition by nucleic acid aptamers

Improved thrombin binding aptamer by incorporation of a single unlocked nucleic acid monomer

A 15-mer DNA aptamer (named TBA) adopts a G-quadruplex structure that strongly inhibits fibrin-clot formation by binding to thrombin. We have performed thermodynamic analysis, binding affinity and biological activity studies of TBA variants modified by unlocked nucleic acid (UNA) monomers. UNA-U placed in position U3, U7 or U12 increases the thermodynamic stability of TBA by 0.15–0.50 kcal/mol. In contrast, modification of any position within the two G-quartet structural elements is unfavorable for quadruplex formation. The intramolecular folding of the quadruplexes is confirmed by Tm versus ln c analysis. Moreover, circular dichroism and thermal difference spectra of the modified TBAs displaying high thermodynamic stability show bands that are characteristic for antiparallel quadruplex formation. Surface plasmon resonance studies of the binding of the UNA-modified TBAs to thrombin show that a UNA monomer is allowed in many positions of the aptamer without significantly changing the thrombin-binding properties. The biological effect of a selection of the modified aptamers was tested by a thrombin time assay and showed that most of the UNA-modified TBAs possess anticoagulant properties, and that the construct with a UNA-U monomer in position 7 is a highly potent inhibitor of fibrin-clot formation

In vitro initial attachment of HIV-1 integrase to viral ends: control of the DNA specific interaction by the oligomerization state

HIV-1 integrase (IN) oligomerization and DNA recognition are crucial steps for the subsequent events of the integration reaction. Recent advances described the involvement of stable intermediary complexes including dimers and tetramers in the in vitro integration processes, but the initial attachment events and IN positioning on viral ends are not clearly understood. In order to determine the role of the different IN oligomeric complexes in these early steps, we performed in vitro functional analysis comparing IN preparations having different oligomerization properties. We demonstrate that in vitro IN concerted integration activity on a long DNA substrate containing both specific viral and nonspecific DNA sequences is highly dependent on binding of preformed dimers to viral ends. In addition, we show that IN monomers bound to nonspecific DNA can also fold into functionally different oligomeric complexes displaying nonspecific double-strand DNA break activity in contrast to the well known single strand cut catalyzed by associated IN. Our results imply that the efficient formation of the active integration complex highly requires the early correct positioning of monomeric integrase or the direct binding of preformed dimers on the viral ends. Taken together the data indicates that IN oligomerization controls both the enzyme specificity and activity

Broad-spectrum aptamer inhibitors of HIV reverse transcriptase closely mimic natural substrates

A detailed understanding of how aptamers recognize biological binding partners is of considerable importance in the development of oligonucleotide therapeutics. For antiviral nucleic acid aptamers, current models predict a correlation between broad-spectrum inhibition of viral proteins and suppression of emerging viral resistance, but there is little understanding of how aptamer structures contribute to recognition specificity. We previously established that two independent single-stranded DNA aptamers, R1T and RT1t49(−5), are potent inhibitors of reverse transcriptases (RTs) from diverse branches of the primate lentiviral family, including HIV-1, HIV-2 and SIV(cpz). In contrast, class 1 RNA pseudoknots, such as aptamer T1.1, are specific for RTs from only a few viral clades. Here, we map the binding interfaces of complexes formed between RT and aptamers R1T, RT1t49(−5) and T1.1, using mass spectrometry-based protein footprinting of RT and hydroxyl radical footprinting of the aptamers. These complementary methods reveal that the broad-spectrum aptamers make contacts throughout the primer-template binding cleft of RT. The double-stranded stems of these aptamers closely mimic natural substrates near the RNase H domain, while their binding within the polymerase domain significantly differs from RT substrates. These results inform our perspective on how sustained, broad-spectrum inhibition of RT can be achieved by aptamers

Aptamers that recognize drug-resistant HIV-1 reverse transcriptase

Drug-resistant variants of HIV-1 reverse transcriptase (RT) are also known to be resistant to anti-RT RNA aptamers. In order to be able to develop diagnostics and therapies that can focus on otherwise drug-resistant viruses, we have isolated two aptamers against a well-known, drug-resistant HIV-1 RT, Mutant 3 (M3) from the multidrug-resistant HIV-1 RT panel. One aptamer, M302, bound M3 but showed no significant affinity for wild-type (WT) HIV-1 RT, while another aptamer, 12.01, bound to both M3 and WT HIV-1 RTs. In contrast to all previously selected anti-RT aptamers, neither of these aptamers showed observable inhibition of either polymerase or RNase H activities. Aptamers M302 and 12.01 competed with one another for binding to M3, but they did not compete with a pseudoknot aptamer for binding to the template/primer cleft of WT HIV-1 RT. These results represent the surprising identification of an additional RNA-binding epitope on the surface of HIV-1 RT. M3 and WT HIV-1 RTs could be distinguished using an aptamer-based microarray. By probing protein conformation as a correlate to drug resistance we introduce an additional and useful measure for determining HIV-1 drug resistance

Stacking of G-quadruplexes: NMR structure of a G-rich oligonucleotide with potential anti-HIV and anticancer activity†

G-rich oligonucleotides T30695 (or T30923), with the sequence of (GGGT)4, and T40214, with the sequence of (GGGC)4, have been reported to exhibit anti-HIV and anticancer activity. Here we report on the structure of a dimeric G-quadruplex adopted by a derivative of these sequences in K+ solution. It comprises two identical propeller-type parallel-stranded G-quadruplex subunits each containing three G-tetrad layers that are stacked via the 5′-5′ interface. We demonstrated control over the stacking of the two monomeric subunits by sequence modifications. Our analysis of possible structures at the stacking interface provides a general principle for stacking of G-quadruplexes, which could have implications for the assembly and recognition of higher-order G-quadruplex structures

Antibody Complementarity-Determining Regions (CDRs) Can Display Differential Antimicrobial, Antiviral and Antitumor Activities

Background: Complementarity-determining regions (CDRs) are immunoglobulin (Ig) hypervariable domains that determine specific antibody (Ab) binding. We have shown that synthetic CDR-related peptides and many decapeptides spanning the variable region of a recombinant yeast killer toxin-like antiidiotypic Ab are candidacidal in vitro. An alanine-substituted decapeptide from the variable region of this Ab displayed increased cytotoxicity in vitro and/or therapeutic effects in vivo against various bacteria, fungi, protozoa and viruses. the possibility that isolated CDRs, represented by short synthetic peptides, may display antimicrobial, antiviral and antitumor activities irrespective of Ab specificity for a given antigen is addressed here.Methodology/Principal Findings: CDR-based synthetic peptides of murine and human monoclonal Abs directed to: a) a protein epitope of Candida albicans cell wall stress mannoprotein; b) a synthetic peptide containing well-characterized B-cell and T-cell epitopes; c) a carbohydrate blood group A substance, showed differential inhibitory activities in vitro, ex vivo and/or in vivo against C. albicans, HIV-1 and B16F10-Nex2 melanoma cells, conceivably involving different mechanisms of action. Antitumor activities involved peptide-induced caspase-dependent apoptosis. Engineered peptides, obtained by alanine substitution of Ig CDR sequences, and used as surrogates of natural point mutations, showed further differential increased/unaltered/decreased antimicrobial, antiviral and/or antitumor activities. the inhibitory effects observed were largely independent of the specificity of the native Ab and involved chiefly germline encoded CDR1 and CDR2 of light and heavy chains.Conclusions/Significance: the high frequency of bioactive peptides based on CDRs suggests that Ig molecules are sources of an unlimited number of sequences potentially active against infectious agents and tumor cells. the easy production and low cost of small sized synthetic peptides representing Ig CDRs and the possibility of peptide engineering and chemical optimization associated to new delivery mechanisms are expected to give rise to a new generation of therapeutic agents.Department of Education, Universities and Research, Basque GovermentFundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Istituto Superiore di Sanita, National Research Project on A.I.D.S.Cariparma Banking FoundationBrazilian National Research CouncilUniv Parma, Sez Microbiol, Dipartimento Patol, I-43100 Parma, ItalyUniv Basque Country, Fac Med Odontol, Dept Inmunol, Microbiol Parasitol, Bilbao, SpainUniv Basque Country, Dept Enfermeria I, Bilbao, SpainUniv Milan, Dipartimento Sci Cliniche L Sacco, Sez Malattie Infettive Immunopatol, Milan, ItalyUniv Studi Parma, Dipartimento Clin Med, Nefrol Sci Prev, Parma, ItalyUniversidade Federal de São Paulo, Departamento Microbiol, Imunol Parasitol, Unidade Oncol Expt, São Paulo, BrazilUniversidade Federal de São Paulo, Dept Biofis, São Paulo, BrazilUniversidade Federal de São Paulo, Departamento Microbiol, Imunol Parasitol, Unidade Oncol Expt, São Paulo, BrazilUniversidade Federal de São Paulo, Dept Biofis, São Paulo, BrazilDepartment of Education, Universities and Research, Basque Goverment: IT-264-07FAPESP: 06/50634-2Istituto Superiore di Sanita, National Research Project on A.I.D.S.: 50G.30Istituto Superiore di Sanita, National Research Project on A.I.D.S.: 40D.14Cariparma Banking Foundation: 2004.0190Brazilian National Research Council: research fellowshipWeb of Scienc

Novel therapeutic strategies targeting HIV integrase

Integration of the viral genome into host cell chromatin is a pivotal and unique step in the replication cycle of retroviruses, including HIV. Inhibiting HIV replication by specifically blocking the viral integrase enzyme that mediates this step is an obvious and attractive therapeutic strategy. After concerted efforts, the first viable integrase inhibitors were developed in the early 2000s, ultimately leading to the clinical licensure of the first integrase strand transfer inhibitor, raltegravir. Similarly structured compounds and derivative second generation integrase strand transfer inhibitors, such as elvitegravir and dolutegravir, are now in various stages of clinical development. Furthermore, other mechanisms aimed at the inhibition of viral integration are being explored in numerous preclinical studies, which include inhibition of 3' processing and chromatin targeting. The development of new clinically useful compounds will be aided by the characterization of the retroviral intasome crystal structure. This review considers the history of the clinical development of HIV integrase inhibitors, the development of antiviral drug resistance and the need for new antiviral compounds