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One-step selection of Vaccinia virus-binding DNA aptamers by MonoLEX.

Nitsche A, Kurth A, Dunkhorst A, Pänke O, Sielaff H, Junge W, Muth D, Scheller F, Stöcklein W, Dahmen C, Pauli G, Kage A - BMC Biotechnol. (2007)

Bottom Line: Most aptamers were isolated from complex libraries of synthetic nucleic acids by a process termed SELEX based on several selection and amplification steps.MonoLEX comprises a single affinity chromatography step, followed by subsequent physical segmentation of the affinity resin and a single final PCR amplification step of bound aptamers.Therefore, this procedure improves the selection of high affinity aptamers by reducing the competition between aptamers of different affinities during the PCR step, indicating an advantage for the single-round MonoLEX method.

View Article: PubMed Central - HTML - PubMed

Affiliation: Centre for Biological Safety 1, Robert Koch-Institut, Nordufer 20, 13353 Berlin, Germany. nitsche@rki.de

ABSTRACT

Background: As a new class of therapeutic and diagnostic reagents, more than fifteen years ago RNA and DNA aptamers were identified as binding molecules to numerous small compounds, proteins and rarely even to complete pathogen particles. Most aptamers were isolated from complex libraries of synthetic nucleic acids by a process termed SELEX based on several selection and amplification steps. Here we report the application of a new one-step selection method (MonoLEX) to acquire high-affinity DNA aptamers binding Vaccinia virus used as a model organism for complex target structures.

Results: The selection against complete Vaccinia virus particles resulted in a 64-base DNA aptamer specifically binding to orthopoxviruses as validated by dot blot analysis, Surface Plasmon Resonance, Fluorescence Correlation Spectroscopy and real-time PCR, following an aptamer blotting assay. The same oligonucleotide showed the ability to inhibit in vitro infection of Vaccinia virus and other orthopoxviruses in a concentration-dependent manner.

Conclusion: The MonoLEX method is a straightforward procedure as demonstrated here for the identification of a high-affinity DNA aptamer binding Vaccinia virus. MonoLEX comprises a single affinity chromatography step, followed by subsequent physical segmentation of the affinity resin and a single final PCR amplification step of bound aptamers. Therefore, this procedure improves the selection of high affinity aptamers by reducing the competition between aptamers of different affinities during the PCR step, indicating an advantage for the single-round MonoLEX method.

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Antiviral activity of A38 against various OPV. OPV were propagated in cell culture in presence or absence of A38 as described above. The degree of infection in comparison to a positive control in the absence of A38 was determined by enumerating the number of infected cells (IFA, black bars), quantification of intracellular poxvirus DNA by real-time PCR (red bars) or determination of the poxvirus titer in the corresponding supernatant (green bars).
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Figure 5: Antiviral activity of A38 against various OPV. OPV were propagated in cell culture in presence or absence of A38 as described above. The degree of infection in comparison to a positive control in the absence of A38 was determined by enumerating the number of infected cells (IFA, black bars), quantification of intracellular poxvirus DNA by real-time PCR (red bars) or determination of the poxvirus titer in the corresponding supernatant (green bars).

Mentions: The antiviral potency of A38 was measured by an endpoint dilution (5 μM, 2.5 μM, 250 nM, 25 nM, 2.5 nM, 250 pM) and immuno-fluorescence-based assay in Hep2 cells against VACV (Fig. 4), followed by determining the ratio of infected/non-infected cells. A38 inhibited VACV entry with a 50% inhibitory concentration (IC50) value of 0.59 μM. The antiviral effect exerted by A38 was neither due to cytotoxicity nor to induced apoptosis. A38 had no significant effect on cell viability in cytotoxicity assays (WST-1, Roche, Germany). The compound concentration at which uninfected Hep2 cell proliferation was inhibited by 50% (CC50) was > 10 μM, which was above the available synthesized concentration for A38. This resulted in a selective index (SI) of > 16.95 (SI = CC50/IC50). A similar antiviral activity of A38 could be detected against a broad spectrum of OPV-like Cowpox virus, Ectromelia virus and Camelpox virus as shown by IFA, by quantitative real-time PCR of viral DNA used as described previously [26] and by virus titration of the corresponding supernatants (Fig. 5). Reduced virus proliferation could be confirmed in all experiments. Taken together, these results demonstrate that A38 binds to a target common to all OPV, is a potent and specific inhibitor of OPV. However, the inhibition of Ectromelia virus proliferation was less pronounced.


One-step selection of Vaccinia virus-binding DNA aptamers by MonoLEX.

Nitsche A, Kurth A, Dunkhorst A, Pänke O, Sielaff H, Junge W, Muth D, Scheller F, Stöcklein W, Dahmen C, Pauli G, Kage A - BMC Biotechnol. (2007)

Antiviral activity of A38 against various OPV. OPV were propagated in cell culture in presence or absence of A38 as described above. The degree of infection in comparison to a positive control in the absence of A38 was determined by enumerating the number of infected cells (IFA, black bars), quantification of intracellular poxvirus DNA by real-time PCR (red bars) or determination of the poxvirus titer in the corresponding supernatant (green bars).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC1994675&req=5

Figure 5: Antiviral activity of A38 against various OPV. OPV were propagated in cell culture in presence or absence of A38 as described above. The degree of infection in comparison to a positive control in the absence of A38 was determined by enumerating the number of infected cells (IFA, black bars), quantification of intracellular poxvirus DNA by real-time PCR (red bars) or determination of the poxvirus titer in the corresponding supernatant (green bars).
Mentions: The antiviral potency of A38 was measured by an endpoint dilution (5 μM, 2.5 μM, 250 nM, 25 nM, 2.5 nM, 250 pM) and immuno-fluorescence-based assay in Hep2 cells against VACV (Fig. 4), followed by determining the ratio of infected/non-infected cells. A38 inhibited VACV entry with a 50% inhibitory concentration (IC50) value of 0.59 μM. The antiviral effect exerted by A38 was neither due to cytotoxicity nor to induced apoptosis. A38 had no significant effect on cell viability in cytotoxicity assays (WST-1, Roche, Germany). The compound concentration at which uninfected Hep2 cell proliferation was inhibited by 50% (CC50) was > 10 μM, which was above the available synthesized concentration for A38. This resulted in a selective index (SI) of > 16.95 (SI = CC50/IC50). A similar antiviral activity of A38 could be detected against a broad spectrum of OPV-like Cowpox virus, Ectromelia virus and Camelpox virus as shown by IFA, by quantitative real-time PCR of viral DNA used as described previously [26] and by virus titration of the corresponding supernatants (Fig. 5). Reduced virus proliferation could be confirmed in all experiments. Taken together, these results demonstrate that A38 binds to a target common to all OPV, is a potent and specific inhibitor of OPV. However, the inhibition of Ectromelia virus proliferation was less pronounced.

Bottom Line: Most aptamers were isolated from complex libraries of synthetic nucleic acids by a process termed SELEX based on several selection and amplification steps.MonoLEX comprises a single affinity chromatography step, followed by subsequent physical segmentation of the affinity resin and a single final PCR amplification step of bound aptamers.Therefore, this procedure improves the selection of high affinity aptamers by reducing the competition between aptamers of different affinities during the PCR step, indicating an advantage for the single-round MonoLEX method.

View Article: PubMed Central - HTML - PubMed

Affiliation: Centre for Biological Safety 1, Robert Koch-Institut, Nordufer 20, 13353 Berlin, Germany. nitsche@rki.de

ABSTRACT

Background: As a new class of therapeutic and diagnostic reagents, more than fifteen years ago RNA and DNA aptamers were identified as binding molecules to numerous small compounds, proteins and rarely even to complete pathogen particles. Most aptamers were isolated from complex libraries of synthetic nucleic acids by a process termed SELEX based on several selection and amplification steps. Here we report the application of a new one-step selection method (MonoLEX) to acquire high-affinity DNA aptamers binding Vaccinia virus used as a model organism for complex target structures.

Results: The selection against complete Vaccinia virus particles resulted in a 64-base DNA aptamer specifically binding to orthopoxviruses as validated by dot blot analysis, Surface Plasmon Resonance, Fluorescence Correlation Spectroscopy and real-time PCR, following an aptamer blotting assay. The same oligonucleotide showed the ability to inhibit in vitro infection of Vaccinia virus and other orthopoxviruses in a concentration-dependent manner.

Conclusion: The MonoLEX method is a straightforward procedure as demonstrated here for the identification of a high-affinity DNA aptamer binding Vaccinia virus. MonoLEX comprises a single affinity chromatography step, followed by subsequent physical segmentation of the affinity resin and a single final PCR amplification step of bound aptamers. Therefore, this procedure improves the selection of high affinity aptamers by reducing the competition between aptamers of different affinities during the PCR step, indicating an advantage for the single-round MonoLEX method.

Show MeSH
Related in: MedlinePlus