Oligodeoxynucleotides containing conformationally constrained abasic sites: a UV and fluorescence spectroscopic investigation on duplex stability and structure

Abstract

The synthesis and incorporation into oligodeoxy-nucleotides of two novel, conformationally restricted abasic (AB) site analogs are described. The stability of oligonucleotide 18mer duplexes containing one such AB site opposite any of the four natural DNA bases was investigated by UV melting curve analysis and compared to that of duplexes containing a conformationally flexible propanediol unit 1 or a tetrahydrofuran unit 2 as an AB site analog. No major differences in the melting temperatures (DeltaT(m) 0-3 degrees C) between the different abasic duplexes were observed. All AB duplexes were found to have T(m)s that were lower by 9-15 degrees C relative to a fully matched 18mer control duplex, and by 4-10 degrees C relative to the corresponding 19mer duplexes in which the AB site is replaced by a mismatched nucleobase. Thus we conclude that the loss of stability of a duplex that is encountered by removal of a nucleobase from the stack cannot be compensated with conformational restriction of the AB site. From the van't Hoff transition enthalpies obtained from the melting curves, it appears that melting cooperativity is higher for the duplexes containing the conformationally rigid AB sites. Fluorescence quenching experiments with duplexes containing the fluorescent base 2-amino-purine (2AP) opposite the AB sites showed a weak tendency towards more efficient stacking of this base in duplexes containing the conformationally constrained AB sites. Thus, such AB sites may structurally stabilize the cavity formed by the removal of a base. Potential applications emerging from the properties of such conformationally constrained AB sites in DNA diagnostics are discussed.

The Effect of a Non-nucleosidic Phenanthrene Building Block on DNA Duplex Stability

“…To better understand the contribution of the phenanthrene moiety to duplex formation, we synthesized oligonucleotides 19 and 20 containing the linker L, in which a simple CH 2 unit has replaced the phenanthrene. A strong decrease (DT m À 17.78) in duplex stability was observed, when L was placed opposite to a natural base (adenine; see Table 1, Entry 8), which is in agreement with the data observed with a similar linker [12]. A duplex containing two Ls in opposite positions (Entry 9) shows an even larger destabilization (DT m À 20.78).…”

A Stable DNA Duplex Containing a Non-Hydrogen-Bonding and Non-Shape-Complementary Base Couple: Interstrand Stacking as the Stability Determining Factor

“…The incorporation of 7 into the duplex 8 (Table 1) was effected by standard protocols in automated DNA synthesis 25. The thermal stability of the duplexes 8 (X, Y=dBP, dA, dG, dC, dT, and the abasic site analogue ϕ) was assessed by determination of the melting temperatures ( T m ) by using UV spectroscopy (10 m M NaH 2 PO 4 , 150 m M NaCl, pH 7.0; Figure 1 a; Table 1).…”

Site-Specific Variations in RNA Folding Thermodynamics Visualized by 2-Aminopurine Fluorescence

“…The fluorescence properties of 2-AP are extremely sensitive to local changes in conformation, making this base analogue nearly ideal for studies involving structural dynamics (4-7), mismatch (8,9) or abasic (10)(11)(12) sites, base flipping (2,13,14), and protein-nucleic acid association (15)(16)(17)(18). Examples include characterization of ribozyme folding (19)(20)(21) and catalysis (22,23), riboswitches (24), interactions with polymerases (25)(26)(27), and nucleic aciddrug interactions (28)(29)(30)(31).…”