Binding of biotinylated DNA-oligonucleotides to different coagulation factors of the intrinsic coagulation pathway.

<p>Microtiter plate wells were coated with 10 µg/mL each of (A) kininogen or (B) prekallikrein and binding of increasing concentrations of the biotinylated DNA-oligonucleotides 21mer-H1 (closed circles), 21mer-H3 (open triangles), 21mer-L1 (closed squares) was assessed. All data represent mean ± SD (n = 3; *p<0.05; 21mer-L1 and 21mer-H3 vs. 21mer-H1) of one representative experiment out of three independent ones. (C) Microtiter plate wells were coated with 10 µg/mL kininogen, factor XI (FXI) or factor XII (FXII) each and incubated with 25 µg/mL each of different biotinylated DNA-oligonucleotides: 21mer-H1 (black bars), 21mer-H3 (white bars) or 21mer-L (hatched bars). All data represent mean ± SD (n = 3) of one representative experiment out of three independent ones. (D) Increasing concentrations of the biotinylated DNA-oligonucleotides 21mer-H1 (closed circles), 21mer-H3 (open triangles) or 21mer-L1 (closed squares) were analyzed for prekallikrein auto-activation. All data represent mean ± SEM (n≥3; *p<0.05; 21mer-H1 vs. 21mer-H3).</p

Stability of DNA- and RNA-oligonucleotides in human plasma.

<p>(A) Integrity of 21mer-L1 and (B) 21mer-H1 DNA- and RNA-oligonucleotides was confirmed by polyacrylamide gel electrophoresis without or after preincubation in pooled human plasma for 1 or 5 min, respectively. Each panel represents one representative experiment out of three independent ones.</p

Influences of DNA-aptamers on the intrinsic coagulation pathway.

<p>(A) The activation of prekallikrein was followed in the presence of increasing doses of the DNA-aptamers 15mer-thrombin (open circles, interrupted line), 44mer-APC (closed squares), 26mer-AS1411 (closed circles) or 25mer-VEGF (closed triangles, dotted line). (B) Turbidity clot-lysis assays were performed in the absence (black bars) or presence of 1.25 µg/mL (white bars) and 10 µg/mL (striped bars) of the DNA-aptamers 15mer-thrombin, 44mer-APC, 26mer-AS1411 or 25mer-VEGF, respectively. Coagulation was initiated by recalcification, clotting times were defined as respective time points of maximal absorbance. The clotting time of untreated plasma was defined as 100%. All data represent mean ± SEM (n = 3; *p<0.05; 1.25 µg/mL or 10 µg/mL vs. control). (C) The activation of prekallikrein was followed in the presence of increasing doses of the oligonucleotide 21mer-H1 (closed circles) and 21mer-H1-HEG (closed squares). All data represent mean ± SEM (n = 6).</p

Sequences and secondary structures of DNA- and RNA-oligonucleotides.

<p>Described are the secondary structures of (A) DNA- and (B) RNA-oligonucleotides as predicted by the mFold DNA or RNA database. Delta G (ΔG) values represent changes in free enthalpy representative for the stability of the compounds with negative (exergonic) or positive values (endergonic).</p

Procoagulant activity of snRNAs.

<p>(A) Increasing concentrations of U6snRNA (closed triangles, black line) or poly (I:C) (closed diamonds, dotted line) were analyzed for prekallikrein auto-activation. All data represent mean ± SEM (n = 3). (B) Integrity of U6snRNA was confirmed by agarose gelelectrophoresis.</p

Procoagulant activity of different linear and hairpin-forming DNA-oligonucleotides.

<p>Increasing concentrations of the linear DNA-oligonucleotides 21mer-L1 (closed squares), 21mer-L2 (closed triangels) or 21mer-L3 (closed circles) were analyzed for (A) prekallikrein auto-activation or (B) procoagulant activity in a turbidity clot-lysis assay. The clotting time of untreated plasma was defined as 100%. All data represent mean ± SEM (n = 3). Increasing concentrations of the hairpin-forming DNA-oligonucleotides 21mer-H1 (closed circles), 21mer-H2 (open squares) or 21mer-H3 (open triangles) were analyzed for (C) prekallikrein auto-activation or (D) procoagulant activity in a turbidity clot-lysis assay. The clotting time of untreated plasma was defined as 100%. All data represent mean ± SEM (n≥3; *p<0.05; 21mer-H1 vs. 21mer-H3; #p<0.05; 21mer-H2 vs. 21mer-H3). (E) The sizes of DNA-oligonucleotides were analyzed by polyacrylamide gel electrophoresis. Shown is one representative experiment out of three independent ones.</p