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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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Related in: MedlinePlus

Schematic presentation of MonoLEX in comparison to SELEX. Based on a combinatory oligonucleotide library, SELEX comprises several cycles of target binding, elution and amplification of putative aptamers. In contrast, MonoLEX starts with one affinity chromatography to sort non-binding oligonucleotides, low-affinity aptamers and high-affinity aptamers. Highly affine aptamers are amplified once and characterized further by an aptamer blot assay.
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Figure 1: Schematic presentation of MonoLEX in comparison to SELEX. Based on a combinatory oligonucleotide library, SELEX comprises several cycles of target binding, elution and amplification of putative aptamers. In contrast, MonoLEX starts with one affinity chromatography to sort non-binding oligonucleotides, low-affinity aptamers and high-affinity aptamers. Highly affine aptamers are amplified once and characterized further by an aptamer blot assay.

Mentions: As an alternative to the SELEX process with 7 to more than 30 selection and amplification cycles requiring large amounts of the target molecule, a new selection process was established. Based on reports about selecting functional oligonucleotides [23] and the potential of oligonucleotides as non-Watson-Crick-type binders to peptides and proteins [24], the present study applied this new one-step aptamer isolation protocol (MonoLEX) to retrieve DNA aptamers that have the potential to bind specifically to OPV particles. The MonoLEX approach combined a single affinity chromatography step with subsequent physical segmentation of the affinity resin and one single final exponential amplification step of bound aptamers. A schematic comparison of SELEX and MonoLEX is given in figure 1. Specific aptamers were selected from a combinatory library of oligonucleotides characterized by two flanking primers of known sequence and an internal region of 20 random nucleotide positions (N20 DNA library). Under adequate chromatographic conditions, like flow laminarity, sufficient capacity and homogeneity of the resin, high-affinity-binding aptamers stuck to the target, whereas weakly binding oligonucleotides could be removed from the resin by ample washing. Since the selection is an in vitro process, depending on the final application of the aptamer, the selection conditions can be adapted accordingly.


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)

Schematic presentation of MonoLEX in comparison to SELEX. Based on a combinatory oligonucleotide library, SELEX comprises several cycles of target binding, elution and amplification of putative aptamers. In contrast, MonoLEX starts with one affinity chromatography to sort non-binding oligonucleotides, low-affinity aptamers and high-affinity aptamers. Highly affine aptamers are amplified once and characterized further by an aptamer blot assay.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: Schematic presentation of MonoLEX in comparison to SELEX. Based on a combinatory oligonucleotide library, SELEX comprises several cycles of target binding, elution and amplification of putative aptamers. In contrast, MonoLEX starts with one affinity chromatography to sort non-binding oligonucleotides, low-affinity aptamers and high-affinity aptamers. Highly affine aptamers are amplified once and characterized further by an aptamer blot assay.
Mentions: As an alternative to the SELEX process with 7 to more than 30 selection and amplification cycles requiring large amounts of the target molecule, a new selection process was established. Based on reports about selecting functional oligonucleotides [23] and the potential of oligonucleotides as non-Watson-Crick-type binders to peptides and proteins [24], the present study applied this new one-step aptamer isolation protocol (MonoLEX) to retrieve DNA aptamers that have the potential to bind specifically to OPV particles. The MonoLEX approach combined a single affinity chromatography step with subsequent physical segmentation of the affinity resin and one single final exponential amplification step of bound aptamers. A schematic comparison of SELEX and MonoLEX is given in figure 1. Specific aptamers were selected from a combinatory library of oligonucleotides characterized by two flanking primers of known sequence and an internal region of 20 random nucleotide positions (N20 DNA library). Under adequate chromatographic conditions, like flow laminarity, sufficient capacity and homogeneity of the resin, high-affinity-binding aptamers stuck to the target, whereas weakly binding oligonucleotides could be removed from the resin by ample washing. Since the selection is an in vitro process, depending on the final application of the aptamer, the selection conditions can be adapted accordingly.

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