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


CONCLUSIONS
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.
Nitsche
Andreas
A
Centre for Biological Safety 1, Robert Koch-Institut, Nordufer 20, 13353 Berlin, Germany. nitsche@rki.de
Kurth
Andreas
A
Dunkhorst
Anna
A
Pänke
Oliver
O
Sielaff
Hendrik
H
Junge
Wolfgang
W
Muth
Doreen
D
Scheller
Frieder
F
Stöcklein
Walter
W
Dahmen
Claudia
C
Pauli
Georg
G
Kage
Andreas
A
D052157:Aptamers, Nucleotide / Q000235:genetics* / Q000302:isolation & purification*; D002846:Chromatography, Affinity / Q000379:methods*; D004279:DNA, Viral / Q000235:genetics* / Q000302:isolation & purification*; D018390:Gene Targeting / Q000379:methods*; D016133:Polymerase Chain Reaction / Q000379:methods; D014616:Vaccinia virus / Q000235:genetics*
D052157:Aptamers, Nucleotide; D004279:DNA, Viral
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