An RNA Aptamer That Discriminates Bovine Factor IX from Human Factor IX

Gopinath, Subash C.B.; Dhakshnamoorthy, Balasundaresan; Akitomi, Joe; Mizuno, Hiroshi

doi:10.1093/jb/mvj203

Cited by 35 publications

(20 citation statements)

References 21 publications

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“…Aptamers that can discriminate between closely related species are extremely useful and aid in the development of specific diagnostic reagents. In the past, several aptamers were reported to have high abilities to discriminate between closely related molecules and species with several-thousandfold discriminatory abilities (10,13,17). Because the ectodomains of the gD proteins of HSV-1 and HSV-2 share 86% sequence identity, we were interested in assessing the interactions of the selected aptamers with gDs derived from HSV-1 and HSV-2.…”

Section: Resultsmentioning

confidence: 99%

Aptamer That Binds to the gD Protein of Herpes Simplex Virus 1 and Efficiently Inhibits Viral Entry

Gopinath

Hayashi

Kumar

2012

J Virol

103

107

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The ectodomain of the gD protein of herpes simplex viruses (HSVs) plays an important role in viral entry by binding to specific cellular coreceptors and mediating viral entry to the host cells. In the present study, we isolated RNA aptamers (aptamer-1 and aptamer-5) that specifically bind to the gD protein of HSV-1 with high affinity and are able to discriminate the gD protein of a different virus, HSV-2. Aptamer-1 efficiently interfered with the interaction between the gD protein and the HSV-1 target cell receptor (HVEM) in a dose-dependent manner. The 50% effective concentration (EC 50 ) of aptamer-1 was estimated to be in the nanomolar range (60 nM). Furthermore, aptamer-1 was analyzed for anti-HSV-1 activity by using plaque assays, and it efficiently inhibited viral entry with an estimated K i of 0.8 M. To expand the future applications of aptamer-1, a shorter variant was designed by using both mapping and boundary analyses, resulting in the mini-1 aptamer (44-mer). Compared to the fulllength aptamer, mini-1 had at least as high an affinity, specificity, and ability to interfere with gD-HVEM interactions. These studies suggest that the mini-1 aptamer could be explored further as an anti-HSV-1 topical therapy designed to prevent the risk of acquiring HSV-1 infection through physical contact. Herpes simplex virus 1 (HSV-1) and HSV-2 are widespread human pathogens that infect primarily epithelial tissues before spreading to the nervous system, where they become latent. The genomes of HSV-1 and HSV-2 share 50 to 70% homology, and the two viruses also share several cross-reactive epitopes. The entry of HSV into the host cell begins with the binding of viral proteins gC and gB to proteoglycans on the host cell surface. This binding interaction is then followed by the coordinated action of four essential glycoproteins: gD, gB, gH, and gL (3, 30). Of these four glycoproteins, the gD protein is required for binding to the specific cellular receptors for viral entry. The gD protein recognizes two protein receptors, herpesvirus entry mediator (HVEM) (34) and nectin-1 (25). In addition to these receptor proteins, HSV-1 entry can also be mediated by 3-O-sulfated-modified heparan sulfate (29). The gD proteins of HSV-1 and HSV-2 are highly homologous proteins, with a sequence identity of around 86% within the ectodomain region (amino acids [aa] 1 to 319).The structures of the gD protein of HSV-1 have been solved, both in the absence (Fig. 1a) (4) and in the presence (Fig. 1b) (20) of its cognate receptor, HVEM. Recently, the structure of gD bound to another receptor, nectin-1, was also determined by two groups (Fig. 1c and d) (8, 35). Those studies revealed that gD contains a V-like Ig fold between residues 56 and 184. This structural feature is commonly found in cell surface adhesion molecules. N-and C-terminal extensions flank this fold and are unfolded and disordered in the absence of the gD receptor. However, in the HVEM-bound form, the N terminus of gD folds into a hairpin structure that binds to HVEM (4). This recept...

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Section: Resultsmentioning

confidence: 99%

Aptamer That Binds to the gD Protein of Herpes Simplex Virus 1 and Efficiently Inhibits Viral Entry

Gopinath

Hayashi

Kumar

2012

J Virol

103

107

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“…Following the incubation of the library with the target molecule, those sequences that bind to the target are partitioned from those that do not by numerous methods such as nitrocellulose filter membrane (Gopinath et al 2005(Gopinath et al , 2006a, column matrices (Skrypina et al 2004 andBrockstedt et al 2004), capillary electrophoresis (Mendonsa and Bowser 2004;Schou and Heegaard 2006) and other methods of separation (Gopinath 2007). The retained ligands are then amplified to enrich the target bound ligand molecules and these processes of selection and amplification are reiterated until the ligands with the highest binding affinity are obtained.…”

Section: Introductionmentioning

confidence: 99%

Conditions optimized for the preparation of single-stranded DNA (ssDNA) employing lambda exonuclease digestion in generating DNA aptamer

Citartan

Tang

Tan

et al. 2010

World J Microbiol Biotechnol

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The generation of DNA aptamer by Systematic Evolution of Ligands by Exponential Enrichment requires a good method of ssDNA generation. There are various methods developed to generate ssDNA such as streptavidin-biotin based separation techniques, asymmetric PCR and strand separation of the PCR product containing primer with a terminator and an extension of 20 nucleotides on denaturing urea-polyacrylamide gel. In the present investigation, we have shown the possible improvements for the regular lambda nuclease digestion under optimized conditions. Optimization of the PCR cycles, time course studies on lambda nuclease digestion and purification of the ssDNA from the lambda exonuclease digestion mixture was found to be able to recover ssDNA amounting up to 39.19 ± 2.48 % of the starting amount of dsDNA. These strategies can be applied to the techniques involving essential usage of ssDNA.

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“…However, in this respect, 9.3t is different, apparently being able to inhibit FIXa from humans, pigs and mice. Another RNA aptamer, isolated by library screening with bovine FIX, bound bovine FIX with a K D of ~10 nM but did not measurably bind human FIX (58). The development of the 9.3t aptamer and others have been accompanied by the development of antidotes for rapid reversal of aptamer anticoagulatory effects in the form of complementary oligonucleotides (57,60) or nucleic acid binding polymers (61).…”

Section: General Properties Of Serine Proteases -mentioning

confidence: 99%

“…Aptamers can be conformation-specific as exemplified by thrombin aptamer HD-22 and a PSA aptamer recognizing the active form but not the zymogen form of their targets (39,82). They can also have speciesspecificity as found for bovine FIX aptamers preventing bovine but not measurably human thrombin-catalyzed fibrinogen conversion (58). Likewise, uPA-uPAR blocking uPA aptamers were able to inhibit the interaction in a human but not a mouse setting (72).…”

Section: General Features Concerning Protease-binding Aptamersmentioning

confidence: 99%

Nucleic Acid Aptamers Against Proteases

Dupont

Andersen²,

Bøtkjær³

et al. 2011

CMC

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Proteases are potential or realized therapeutic targets in a wide variety of pathological conditions. Moreover, proteases are classical subjects for studies of enzymatic and regulatory mechanisms. We here review the literature on nucleic acid aptamers selected with proteases as targets. Designing small molecule protease inhibitors of sufficient specificity has proved a daunting task. Aptamers seem to represent a promising alternative. In our review, we concentrate on biochemical mechanisms of aptamer selection, proteinaptamer recognition, protease inhibition, and advantages of aptamers for pharmacological intervention with pathophysiological functions of proteases. Aptamers can be selected so that they bind their targets highly specifically and with affinities corresponding to K D values in the nM range. Aptamers can be selected so that they recognize their targets conformation-specifically, for instance with vastly different affinities to zymogen and active enzyme forms. Aptamers can be selected so that they inhibit the enzyme activity of the target proteases, but also so that they inhibit functionally important exosite interactions, for instance cofactor binding. Several protease inhibiting aptamers, mostly directed against blood coagulation, are in clinical trials as antithrombotic drugs. Several of the studies on protease-binding aptamers have been pioneering and trend-setting in the field. The work with protease-binding aptamers also demonstrates many interesting examples of non-standard selection strategies and of new principles for regulating the activity of the inhibitory action of aptamers of general interest to researchers working with nucleic acid aptamers.

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An RNA Aptamer That Discriminates Bovine Factor IX from Human Factor IX

Cited by 35 publications

References 21 publications

Aptamer That Binds to the gD Protein of Herpes Simplex Virus 1 and Efficiently Inhibits Viral Entry

Aptamer That Binds to the gD Protein of Herpes Simplex Virus 1 and Efficiently Inhibits Viral Entry

Conditions optimized for the preparation of single-stranded DNA (ssDNA) employing lambda exonuclease digestion in generating DNA aptamer

Nucleic Acid Aptamers Against Proteases

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