Synthesis and biophysical properties of C5-functionalized LNA (locked nucleic acid).
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's 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'-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'-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.
Department of Chemistry, University of Idaho , Moscow, Idaho 83844-2343, United States.
Pawan
P
Kumar
Michael E
ME
Østergaard
Bharat
B
Baral
Brooke A
BA
Anderson
Dale C
DC
Guenther
Mamta
M
Kaura
Daniel J
DJ
Raible
Pawan K
PK
Sharma
Patrick J
PJ
Hrdlicka
D008968:Molecular Conformation; D009690:Nucleic Acid Conformation; D009696:Nucleic Acids / Q000737:chemistry*; D009838:Oligodeoxyribonucleotides / Q000737:chemistry*; D009841:Oligonucleotides / Q000737:chemistry*; D009943:Organophosphorus Compounds / Q000138:chemical synthesis* / Q000737:chemistry*; D012313:RNA / Q000737:chemistry*; D014529:Uridine / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000737:chemistry
D009696:Nucleic Acids; D009838:Oligodeoxyribonucleotides; D009841:Oligonucleotides; D009943:Organophosphorus Compounds; C477371:locked nucleic acid; C101323:uridine phosphoramidite; D012313:RNA; D014529:Uridine