Selection of DNA aptamers against rat liver X receptors.
Liver X receptors alpha and beta (LXRalpha; LXRbeta) are members of the nuclear hormone receptor superfamily of ligand-activated transcription factors. LXRs play an important role in the reverse cholesterol transport and govern the expression of many of the proteins that are indispensable for the regulation of normal cholesterol levels in the body. SELEX, an in vitro selection technology, was used on a single stranded DNA library harboring a 12 randomized nucleotide sequence in order to isolate aptamers showing affinity for LXRalpha. Enzyme-linked assays and surface plasmon resonance measurements showed that the selected aptamers had strong affinities for LXRalpha with apparent dissociation constants, K(d)s, in nanomolar range. All clones carried CG-repeats, indicating a probability for a similar manner of binding to LXRalpha. Very high cross-reactivities were observed when testing the aptamers with LXRbeta (up to 700%) and RXRalpha (up to 50%). If instead we regard the aptamer sequences as selected against LXRbeta, the cross-reactivities decrease considerably, to 17% for LXRalpha and 7% for RXRalpha. Therefore, in the future we are planning to use the obtained aptamers as binders for LXRbeta.
Surugiu-Wärnmark
Ioana
I
Department of Pure and Applied Biochemistry, Center for Chemistry and Chemical Engineering, Lund University, SE-221 00 Lund, Sweden. Ioana.Warnmark@tbiokem.lth.se
Wärnmark
Anette
A
Toresson
Gudrun
G
Gustafsson
Jan-Ake
JA
Bülow
Leif
L
D000818:Animals; D001665:Binding Sites / Q000235:genetics; D003001:Cloning, Molecular / Q000379:methods*; D004247:DNA / Q000737:chemistry* / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics*; D008024:Ligands; D008099:Liver / Q000378:metabolism*; D000071518:Liver X Receptors; D057093:Orphan Nuclear Receptors; D011485:Protein Binding; D051381:Rats; D018160:Receptors, Cytoplasmic and Nuclear / Q000737:chemistry* / Q000235:genetics*; D017422:Sequence Analysis, DNA
D004268:DNA-Binding Proteins; D008024:Ligands; D000071518:Liver X Receptors; C000605215:Nr1h3 protein, rat; D057093:Orphan Nuclear Receptors; D018160:Receptors, Cytoplasmic and Nuclear; D004247:DNA