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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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In vitro-binding studies of A38. (a) Dot blot of VACV (VR-1536), non-infective cell culture supernatant (CC) and CMV, followed by incubation of biotin-conjugated A38 in various concentrations and labeling with a streptavidin peroxidase conjugate. (b) Surface Plasmon Resonance measurements (Biacore) of A38. Sequence of injections (injection start): A38 (150 s); VACV (1050 s); 50 mM sodium carbonate (2320 s); 1:10 diluted VACV (4080 s). The overlaid VACV net binding curves are shown on the right hand side. (c) Fluorescence Correlation Spectroscopy (FCS). Normalized autocorrelation functions (ACF) of tetramethylrhodamine isothiocyanate (TMR): free in solution (black), coupled to A38 alone (blue), coupled to A38 with added CMV (brown), coupled to A38 with added VACV (red), and directly coupled to VACV (green). Control experiments with CMV (brown) and 200-nm latex beads (not shown) mixed with TMR-conjugated A38 showed identical diffusion times to those for TMR-labeled A38 alone, indicating a binding exclusively to VACV particles.
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Figure 3: In vitro-binding studies of A38. (a) Dot blot of VACV (VR-1536), non-infective cell culture supernatant (CC) and CMV, followed by incubation of biotin-conjugated A38 in various concentrations and labeling with a streptavidin peroxidase conjugate. (b) Surface Plasmon Resonance measurements (Biacore) of A38. Sequence of injections (injection start): A38 (150 s); VACV (1050 s); 50 mM sodium carbonate (2320 s); 1:10 diluted VACV (4080 s). The overlaid VACV net binding curves are shown on the right hand side. (c) Fluorescence Correlation Spectroscopy (FCS). Normalized autocorrelation functions (ACF) of tetramethylrhodamine isothiocyanate (TMR): free in solution (black), coupled to A38 alone (blue), coupled to A38 with added CMV (brown), coupled to A38 with added VACV (red), and directly coupled to VACV (green). Control experiments with CMV (brown) and 200-nm latex beads (not shown) mixed with TMR-conjugated A38 showed identical diffusion times to those for TMR-labeled A38 alone, indicating a binding exclusively to VACV particles.

Mentions: Binding characteristics of A38 to VACV were further validated by dot blot analysis of immobilized VACV particles stained with biotinylated A38 (OligoService, Berlin, Germany). As shown in Fig. 3a, A38 binds exclusively to VACV particles and not to the non-infected corresponding cell culture supernatant or Cytomegalovirus (CMV) particles. Even in high concentrations of 10 nM aptamer, binding is still specific for VACV. The binding to VACV particles was concentration dependent and could no longer be observed for A38 concentrations below 10 pM.


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)

In vitro-binding studies of A38. (a) Dot blot of VACV (VR-1536), non-infective cell culture supernatant (CC) and CMV, followed by incubation of biotin-conjugated A38 in various concentrations and labeling with a streptavidin peroxidase conjugate. (b) Surface Plasmon Resonance measurements (Biacore) of A38. Sequence of injections (injection start): A38 (150 s); VACV (1050 s); 50 mM sodium carbonate (2320 s); 1:10 diluted VACV (4080 s). The overlaid VACV net binding curves are shown on the right hand side. (c) Fluorescence Correlation Spectroscopy (FCS). Normalized autocorrelation functions (ACF) of tetramethylrhodamine isothiocyanate (TMR): free in solution (black), coupled to A38 alone (blue), coupled to A38 with added CMV (brown), coupled to A38 with added VACV (red), and directly coupled to VACV (green). Control experiments with CMV (brown) and 200-nm latex beads (not shown) mixed with TMR-conjugated A38 showed identical diffusion times to those for TMR-labeled A38 alone, indicating a binding exclusively to VACV particles.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: In vitro-binding studies of A38. (a) Dot blot of VACV (VR-1536), non-infective cell culture supernatant (CC) and CMV, followed by incubation of biotin-conjugated A38 in various concentrations and labeling with a streptavidin peroxidase conjugate. (b) Surface Plasmon Resonance measurements (Biacore) of A38. Sequence of injections (injection start): A38 (150 s); VACV (1050 s); 50 mM sodium carbonate (2320 s); 1:10 diluted VACV (4080 s). The overlaid VACV net binding curves are shown on the right hand side. (c) Fluorescence Correlation Spectroscopy (FCS). Normalized autocorrelation functions (ACF) of tetramethylrhodamine isothiocyanate (TMR): free in solution (black), coupled to A38 alone (blue), coupled to A38 with added CMV (brown), coupled to A38 with added VACV (red), and directly coupled to VACV (green). Control experiments with CMV (brown) and 200-nm latex beads (not shown) mixed with TMR-conjugated A38 showed identical diffusion times to those for TMR-labeled A38 alone, indicating a binding exclusively to VACV particles.
Mentions: Binding characteristics of A38 to VACV were further validated by dot blot analysis of immobilized VACV particles stained with biotinylated A38 (OligoService, Berlin, Germany). As shown in Fig. 3a, A38 binds exclusively to VACV particles and not to the non-infected corresponding cell culture supernatant or Cytomegalovirus (CMV) particles. Even in high concentrations of 10 nM aptamer, binding is still specific for VACV. The binding to VACV particles was concentration dependent and could no longer be observed for A38 concentrations below 10 pM.

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