Oligonucleotides that are 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 design LNA derivatives that induce even higher binding affinity and specificity, greater enzymatic stability and more desirable pharmacokinetic characteristics. Most of this work has focused on modifications of LNA's oxymethylene bridge. Here, we describe a different approach toward modulating the properties of LNA, i.e., through functionalization of LNA nucleobases. More than twenty structurally diverse nucleobase-functionalized LNA monomers have been synthesized and incorporated into oligodeoxyribonucleotides (ONs), which were then characterized with respect to thermal denaturation, enzymatic stability and fluorescence properties. ONs modified with C5-functionalized LNA pyrimidines that are conjugated to small alkynes display significantly increased affinity toward DNA/RNA targets, improved mismatch discrimination and markedly increased protection against 3'-exonucleases relative to conventional LNA. In contrast, ONs modified with monomers that are conjugated to bulky hydrophobic alkynes display significantly lower affinity toward DNA/RNA targets but also much greater resistance against 3'-exonucleases. ONs modified with C5-fluorophore-functionalized LNA uridine monomers enable excellent fluorescent discrimination of targets with single nucleotide polymorphisms (SNPs). This concept is successfully extended to alpha-L-LNA and C8-functionalized LNA purines. We therefore anticipate that functionalization of nucleobase-functionalization will serve as a general and synthetically straightforward approach for modulation of pharmocodynamic and pharmacokinetic properties of oligonucleotides modified with LNA or other conformationally restricted monomers.

Synthesis and biophysical properties of nucleobase-functionalized LNA (locked nucleic acid)

248th National Meeting (2014)

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