Cell Surface Targeting 8/21/06. Adaptamers A20 A35 A50 Link-aptamer T35 S35 S50 T20 S20 T50 A15.

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Cell Surface Targeting 8/21/06

Adaptamers A20 A35 A50 Link-aptamer T35 S35 S50 T20 S20 T50 A15

Outline Protein coupling to beads The new adaptamers Fluorescence Assay Western Blot *New plans*

Protein Coupling to Beads Coupled aminomethylfluorescein to carboxylated polystyrene beads

Coupled thrombin bead performance LaneCondition L10bp ladder 1Thrombin beads + T0 wash 2 Thrombin beads + T0 elution 3Thrombin beads + S0 wash 4 Thrombin beads + S0 elution 5Blank beads + T0 wash 6Blank beads + T0 elution L

New adaptamer coupling LaneCondition L10bp ladder 120 pmol T pmol S pmol A pmol T pmol S pmol A pmol T pmol S pmol A50 L

New Assay to detect thrombin Magnetic streptavidin beads thrombin adaptamer Wash Anti-thrombin 1 o antibody Shake Secondary antibody Infrared imager Li-Cor Shake Wash Plate

Success! Adaptamer = A15 Control: no thrombin Primary and secondary antibodies added together Control: beads lack streptavidin 2X thrombin General setup: Streptavidin beads + A35 + thrombin

Quantitative results

Western Blot thrombin adaptamer WashShake Agarose streptavidin beads Elute with free streptavidin SDS-PAGE Blot, apply antibodies Infrared imager Li-Cor Invitrogen

Conditions General setup: Streptavidin agarose beads + A20 + thrombin Other conditions: A50 instead of A20, double the amount of A20, thrombin Controls: No adaptamer added; thrombin agarose beads instead of streptavidin beads

DNA LaneCondition L10bp ladder 1A20 Wash 2A20 Elution 32X Wash 42X Elution 5A50 Wash 6A50 Elution 7ND Wash 8ND Elution 9TB Wash 10TB Elution A20: general setup = Streptavidin agarose beads + A20 + thrombin 2X: 2X thrombin, A20 A50: A50 instead of A20 ND: No DNA added TB: Thrombin beads replace streptavidin beads L

Protein L LaneCondition LSeeBlue Plus2 ladder 140 pmol thrombin 2A20 Wash 3A20 Elution 42X Wash 52X Elution 6A50 Wash 7A50 Elution 8ND Wash 9ND Elution 10TB Wash 11TB Elution A20: general setup = Streptavidin agarose beads + A20 + thrombin 2X: 2X thrombin, A20 A50: A50 instead of A20 ND: No DNA added TB: Thrombin beads replace streptavidin beads

Ponceau Red L LaneCondition L SeeBlue Plus2 ladder 140 pmol thrombin 2A20 Wash 3A20 Elution 42X Wash 52X Elution 6A50 Wash 7A50 Elution 8ND Wash 9ND Elution 10TB Wash 11TB Elution A20: general setup = Streptavidin agarose beads + A20 + thrombin 2X: 2X thrombin, A20 A50: A50 instead of A20 ND: No DNA added TB: Thrombin beads replace streptavidin beads

2 weeks for the following: New adaptamer that binds a cell surface –Interchangeable parts; proof of concept –DNA candidates: glioblastoma cells (tenascin- C), red blood cells, leukocytes (L-selectin) –Leukocyte-thrombin? –RNA candidates possible, too. Vary the linking region Strand displacement