Efficient Stabilization of Phosphodiester (PO), Phosphorothioate (PS), and 2鈥?O-Methoxy (2鈥?OMe) DNA路RNA Hybrid Duplexes by Amino Sugars

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• 作者：I. Charles ; Erik Davis ; Dev P. Arya
• 刊名：Biochemistry
• 出版年：2012
• 出版时间：July 10, 2012
• 年：2012
• 卷：51
• 期：27
• 页码：5496-5505
• 全文大小：513K
• 年卷期：v.51,no.27(July 10, 2012)
• ISSN：1520-4995

文摘

Antisense strategies that target DNA路RNA hybrid structures offer potential for the development of new therapeutic drugs. The 伪-sarcin loop region of the 16S rRNA domain has been shown to be a high value target for such strategies. Herein, aminoglycoside interaction with three RNA路DNA 伪-sarcin targeted duplexes (rR路dY, rR路S-dY, and rR路2鈥睴Me-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路dY and phophorothioate modified rR路S-dY duplexes. (3) The rR路2鈥睴Me-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路dY and rR路S-dY duplexes as well as a more pronounced A-form signal for the rR路2鈥睴Me-rY duplex. (6) FID assays paralleled the CD results, yielding similar affinity values between the rR路dY and rR路S-dY duplexes and higher affinities with the rR路2鈥睴Me-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路dY and rR路S-dY duplexes with rR路dY having a K₁ of (1.03 卤 0.58) 脳 10p>7p> Mp>鈥?p> and K₂ of (1.13 卤 0.07) 脳 10p>5p> Mp>鈥?p> while rR路S-dY produced a K₁ of (1.17 卤 0.54) 脳 10p>7p> Mp>鈥?p> and K₂ of (1.27 卤 0.69) 脳 10p>5p> Mp>鈥?p>. (8) The rR路2鈥睴Me-rY produced a slightly higher binding constant values with a K₁ of (1.25 卤 0.24) 脳 10p>7p> Mp>鈥?p> and K₂ of (3.62 卤 0.18) 脳 10p>5p> Mp>鈥?p>. (9) The 螖T_m-derived K_Tm of 3.81 脳 10p>7p> Mp>鈥?p> for rR路S-dY was in relative agreement with the corresponding K₁ of 1.17 脳 10p>7p> Mp>鈥?p> derived constant from the fitted ITC. These results illustrate that the increased DNA路RNA hybrid duplex stability in the presence of aminoglycosides can help extend the roles of aminoglycosides in designing modified ODNs for targeting RNA.