Synthesis and Biophysical Properties of C5-Functionalized LNA (Locked Nucleic Acid)

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• 作者：Pawan Kumar ; Michael E. 脴stergaard ; Bharat Baral ; Brooke A. Anderson ; Dale C. Guenther ; Mamta Kaura ; Daniel J. Raible ; Pawan K. Sharma ; Patrick J. Hrdlicka
• 刊名：Journal of Organic Chemistry
• 出版年：2014
• 出版时间：June 6, 2014
• 年：2014
• 卷：79
• 期：11
• 页码：5047-5061
• 全文大小：584K
• 年卷期：v.79,no.11(June 6, 2014)
• ISSN：1520-6904

文摘

Oligonucleotides modified with conformationally restricted nucleotides such as locked nucleic acid (LNA) monomers are used extensively in molecular biology and medicinal chemistry to modulate gene expression at the RNA level. Major efforts have been devoted to the design of LNA derivatives that induce even higher binding affinity and specificity, greater enzymatic stability, and more desirable pharmacokinetic profiles. Most of this work has focused on modifications of LNA鈥檚 oxymethylene bridge. Here, we describe an alternative approach for modulation of the properties of LNA: i.e., through functionalization of LNA nucleobases. Twelve structurally diverse C5-functionalized LNA uridine (U) phosphoramidites were synthesized and incorporated into oligodeoxyribonucleotides (ONs), which were then characterized with respect to thermal denaturation, enzymatic stability, and fluorescence properties. ONs modified with monomers that are conjugated to small alkynes display significantly improved target affinity, binding specificity, and protection against 3鈥?exonucleases relative to regular LNA. In contrast, ONs modified with monomers that are conjugated to bulky hydrophobic alkynes display lower target affinity yet much greater 3鈥?exonuclease resistance. ONs modified with C5-fluorophore-functionalized LNA-U monomers enable fluorescent discrimination of targets with single nucleotide polymorphisms (SNPs). In concert, these properties render C5-functionalized LNA as a promising class of building blocks for RNA-targeting applications and nucleic acid diagnostics.