2'-Fluoro-Pyrimidine-Modified RNA Aptamers Specific for Lipopolysaccharide Binding Protein (LBP).

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.

Platination of full length tRNA(Ala) and truncated versions of the acceptor stem and anticodon loop.

Nuclear DNA is a well characterized target for many low molecular metal-based drugs, with cisplatin and related antineoplastic compounds as typical examples. Much less is known concerning to what extent targeting of RNA may influence the activity spectrum of these types of drugs. In a preliminary communication by us (Papsai et al., Dalton Trans., 2006, 3515) we were able to show that the folded, three-dimensionally well defined structure of tRNA(Ala) readily interacts with cisplatin. In the present study we have further analyzed the binding preferences within the preferentially targeted stem region (sMh(Ala)) by modulation of the sequence around the G-U wobble base-pair and the net charge of the 3' and 5' ends. Our data show that the adduct profile is strongly influenced by the presence of the 5' end phosphate group. Further, the adduct formation reaction can be prevented by replacement of the G-U wobble base-pair with the fully complementary G-C pair. To further investigate the influence from local sequence on the platination process, a model of the anticodon region (acMh(Ala)) was also investigated. In the absence of consecutive guanine-residues in the stem- and anticodon regions, preferential platination was found to take place at the terminal AG-site in the stem region. However, after introduction of a GG-pair in the anticodon loop, platination was observed also here. At 37 degrees C, pH 6.3 and C(Pt) = 0.10 mM the rate of platination was determined to be ca. 1 x 10(-4) s(-1), with the most rapid reaction observed for interaction with the anticodon model carrying two adjacent guanines in the single-stranded loop. Together, these data show that platination of RNA is highly sequence- and structure-dependent.

Kinetic preference for interaction of cisplatin with the G-C-rich wobble basepair region in both tRNAAla and MhAla.

The anticancer active complex cisplatin interacts preferentially with the common, G-C rich, wobble base pair region of both tRNA(Ala) and Mh(Ala) in a reaction that at pH 6.3 is rate limited by the acid hydrolysis of the metal complex.