Forty modified versions of a streptavidin-binding aptamer each containing single or multiple LNA or α-l-LNA-substitutions were synthesized and their dissociation constants determined by surface plasmon resonance experiments. Both full-length and truncated versions of the aptamer were studied and compared with the unmodified DNA aptamers. A ∼two-fold improvement in binding affinity was achieved by incorporation of LNA nucleotides in the 3'-part of the stems of the streptavidin-binding aptamer whereas LNA- and α-l-LNA-substitutions in the terminal stem increased the serum stability.