Efficient stabilization of phosphodiester (PO), phosphorothioate (PS), and 2'-O-methoxy (2'-OMe) DNA·RNA hybrid duplexes by amino sugars.
Antisense strategies that target DNA·RNA hybrid structures offer potential for the development of new therapeutic drugs. The α-sarcin loop region of the 23S [corrected] rRNA domain has been shown to be a high value target for such strategies. Herein, aminoglycoside interaction with three RNA·DNA α-sarcin targeted duplexes (rR·dY, rR·S-dY, and rR·2'OMe-rY) have been investigated to determine the overall effect of aminoglycoside interaction on the stability, affinity, and conformation of these hybrid duplexes. To this end, UV thermal denaturation, circular dichroism spectroscopy, fluorescence intercalator displacement, and ITC as well as DSC calorimetry experiments were carried out. The results suggest the following. (1) Of all the aminoglycosides studied, neomycin confers the highest thermal stability on all three hybrid duplexes studied. (2) There is no appreciable difference in aminoglycoside-induced thermal stability between the unmodified rR·dY and phophorothioate modified rR·S-dY duplexes. (3) The rR·2'OMe-rY duplexes thermal stability is slightly less than the other two hybrids. (4) In all three duplexes, aminoglycoside-induced thermal stability decreased as the number of amino groups decreased. (5) CD scans revealed similar spectra for the rR·dY and rR·S-dY duplexes as well as a more pronounced A-form signal for the rR·2'OMe-rY duplex. (6) FID assays paralleled the CD results, yielding similar affinity values between the rR·dY and rR·S-dY duplexes and higher affinities with the rR·2'OMe-rY duplex. (7) The overall affinity trend between aminoglycosides and the three duplexes was determined to be neomycin > paromomycin > neamine > ribostamycin. (8) ITC K(a) values revealed similar binding constants for the rR·dY and rR·S-dY duplexes with rR·dY having a K(1) of (1.03 ± 0.58) × 10(7) M(-1) and K(2) of (1.13 ± 0.07) × 10(5) M(-1) while rR·S-dY produced a K(1) of (1.17 ± 0.54) × 10(7) M(-1) and K(2) of (1.27 ± 0.69) × 10(5) M(-1). (8) The rR·2'OMe-rY produced a slightly higher binding constant values with a K(1) of (1.25 ± 0.24) × 10(7) M(-1) and K(2) of (3.62 ± 0.18) × 10(5) M(-1). (9) The ΔT(m)-derived K(Tm) of 3.81 × 10(7) M(-1) for rR·S-dY was in relative agreement with the corresponding K(1) of 1.17 × 10(7) M(-1) derived constant from the fitted ITC. These results illustrate that the increased DNA·RNA hybrid duplex stability in the presence of aminoglycosides can help extend the roles of aminoglycosides in designing modified ODNs for targeting RNA.
Laboratory of Medicinal Chemistry, Department of Chemistry, Clemson University, South Carolina 29634, United States.
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Charles
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Arya
D000617:Aminoglycosides / Q000378:metabolism / Q000494:pharmacology*; D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D001483:Base Sequence; D040761:Biomimetic Materials / Q000737:chemistry; D004247:DNA / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004722:Endoribonucleases / Q000737:chemistry; D005656:Fungal Proteins / Q000737:chemistry; D008958:Models, Molecular; D009690:Nucleic Acid Conformation / Q000187:drug effects; D009691:Nucleic Acid Denaturation / Q000187:drug effects; D009693:Nucleic Acid Hybridization / Q000187:drug effects; D054735:Phosphorothioate Oligonucleotides / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D012313:RNA / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D013696:Temperature; D013816:Thermodynamics
D000617:Aminoglycosides; D000900:Anti-Bacterial Agents; D005656:Fungal Proteins; D054735:Phosphorothioate Oligonucleotides; C014801:alpha-sarcin; D012313:RNA; D004247:DNA; D004722:Endoribonucleases