Streptavidin-coated magnetic beads for DNA strand separation implicate a multitude of problems during cell-SELEX.
Using whole living cells as a target for SELEX (systematic evolution of ligands by exponential enrichment) experiments represents a promising method to generate cell receptor-specific aptamers. These aptamers have a huge potential in diagnostics, therapeutics, imaging, regenerative medicine, and target validation. During the SELEX for selecting DNA aptamers, one important step is the separation of 2 DNA strands to yield one of the 2 strands as single-stranded DNA aptamer. This is being done routinely by biotin labeling of the complementary DNA strand to the desired aptamer and then separating the DNA strand by using streptavidin-coated magnetic beads. After immobilization of the double-stranded DNA on these magnetic beads and alkaline denaturation, the non-biotinylated strand is being eluted and the biotinylated strand is retarded. Using Western blot analysis, we demonstrated the detachment of covalent-bonded streptavidin from the bead surface after alkaline treatment. The eluates were also contaminated with undesired biotinylated strands. Furthermore, a streptavidin-induced aggregation of target cells was demonstrated by flow cytometry and microscopic methods. Cell-specific enrichment of aptamers was not possible due to clustering and patching effects triggered by streptavidin. Therefore, the use of streptavidin-coated magnetic beads for DNA strand separation should be examined thoroughly, especially for cell-SELEX applications.
Paul
Angela
A
Department of Thoracic, Cardiac, and Vascular Surgery, University Hospital, Tübingen 72076, Germany.
Avci-Adali
Meltem
M
Ziemer
Gerhard
G
Wendel
Hans P
HP
D052157:Aptamers, Nucleotide / Q000737:chemistry*; D001710:Biotin / Q000378:metabolism; D001792:Blood Platelets / Q000737:chemistry; D002460:Cell Line; D004277:DNA, Single-Stranded / Q000737:chemistry / Q000302:isolation & purification*; D052203:Ferrosoferric Oxide; D006801:Humans; D008863:Microspheres; D052156:SELEX Aptamer Technique; D019809:Streptavidin / Q000737:chemistry*
D052157:Aptamers, Nucleotide; D004277:DNA, Single-Stranded; D001710:Biotin; D019809:Streptavidin; D052203:Ferrosoferric Oxide