Lipopolysaccaride binding protein (LBP), a glycosylated acute phase protein, plays an important role in the pathophysiology of sepsis. LBP binds with high affinity to the lipid part of bacterial lipopolysaccaride (LPS). Inhibition of the LPS-LBP interaction or blockage of LBP-mediated transfer of LPS monomers to CD14 may be therapeutical strategies to prevent septic shock. LBP is also of interest as a biomarker to identify septic patients at high risk for death, as LBP levels are elevated during early stages of severe sepsis. As a first step toward such potential applications, we isolated aptamers specific for murine LBP (mLBP) by in vitro selection from a library containing a 60-nucleotide randomized region. Modified RNA pools were transcribed in the presence of 2′-fluoro-modified pyrimidine nucleotides to stabilize transcripts against nuclease degradation. As verified for one aptamer experimentally, the selected aptamers adopt a “three-helix junction” architecture, presenting single-stranded 7-nt (5′-YGCTTCY) or 6-nt (5′-RTTTCY) consensus sequences in their core. The best binder (aptamer A011; Kd of 270 nM for binding to mLBP), characterized in more detail by structure probing and boundary analysis, was demonstrated to bind with high specificity to murine LBP.
Discovering Human RNA Aptamers by Structure-Based Bioinformatics and Genome-Based In Vitro Selection
