title: Structural Basis of Duplex Thermodynamic Stability and Enhanced Nuclease Resistance of 5'-C-Methyl Pyrimidine-Modified Oligonucleotides.
abstract: Although judicious use of chemical modifications has contributed to the success of nucleic acid therapeutics, poor systemic stability remains a major hurdle. The introduction of functional groups around the phosphate backbone can enhance the nuclease resistance of oligonucleotides (ONs). Here, we report the synthesis of enantiomerically pure (R)- and (S)-5'-C-methyl (C5'-Me) substituted nucleosides and their incorporation into ONs. These modifications generally resulted in a decrease in thermal stability of oligonucleotide (ON) duplexes in a manner dependent on the stereoconfiguration at C5' with greater destabilization characteristic of (R)-epimers. Enhanced stability against snake venom phosphodiesterase resulted from modification of the 3'-end of an ON with either (R)- or (S)-C5'-Me nucleotides. The (S)-isomers with different 2'-substituents provided greater resistance against 3'-exonucleases than the corresponding (R)-isomers. Crystal structure analyses of RNA octamers with (R)- or (S)-5'-C-methyl-2'-deoxy-2'-fluorouridine [(R)- or (S)-C5'-Me-2'-FU, respectively] revealed that the stereochemical orientation of the C5'-Me and the steric effects that emanate from the alkyl substitution are the dominant determinants of thermal stability and are likely molecular origins of resistance against nucleases. X-ray and NMR structural analyses showed that the (S)-C5'-Me epimers are spatially and structurally more similar to their natural 5' nonmethylated counterparts than the corresponding (R)-epimers.
authors.affiliation: Alnylam Pharmaceuticals , 300 Third Street, Cambridge, Massachusetts 02142, United States.
authors.forename: Alexander V
authors.initials: AV
authors.lastname: Kel'in
authors.affiliation: Alnylam Pharmaceuticals , 300 Third Street, Cambridge, Massachusetts 02142, United States.
authors.forename: Ivan
authors.initials: I
authors.lastname: Zlatev
authors.affiliation: Department of Biochemistry and Center for Structural Biology, Vanderbilt University, School of Medicine , Nashville, Tennessee 37232, United States.
authors.forename: Joel
authors.initials: J
authors.lastname: Harp
authors.affiliation: Alnylam Pharmaceuticals , 300 Third Street, Cambridge, Massachusetts 02142, United States.
authors.forename: Muthusamy
authors.initials: M
authors.lastname: Jayaraman
authors.affiliation: Alnylam Pharmaceuticals , 300 Third Street, Cambridge, Massachusetts 02142, United States.
authors.forename: Anna
authors.initials: A
authors.lastname: Bisbe
authors.affiliation: Alnylam Pharmaceuticals , 300 Third Street, Cambridge, Massachusetts 02142, United States.
authors.forename: Jonathan
authors.initials: J
authors.lastname: O'Shea
authors.affiliation: Alnylam Pharmaceuticals , 300 Third Street, Cambridge, Massachusetts 02142, United States.
authors.forename: Nate
authors.initials: N
authors.lastname: Taneja
authors.affiliation: Alnylam Pharmaceuticals , 300 Third Street, Cambridge, Massachusetts 02142, United States.
authors.forename: Rajar M
authors.initials: RM
authors.lastname: Manoharan
authors.affiliation: Department of Chemistry and Biochemistry, University of California , Los Angeles, California 90095, United States.
authors.forename: Saeed
authors.initials: S
authors.lastname: Khan
authors.affiliation: Alnylam Pharmaceuticals , 300 Third Street, Cambridge, Massachusetts 02142, United States.
authors.forename: Klaus
authors.initials: K
authors.lastname: Charisse
authors.affiliation: Alnylam Pharmaceuticals , 300 Third Street, Cambridge, Massachusetts 02142, United States.
authors.forename: Martin A
authors.initials: MA
authors.lastname: Maier
authors.affiliation: Department of Biochemistry and Center for Structural Biology, Vanderbilt University, School of Medicine , Nashville, Tennessee 37232, United States.
authors.forename: Martin
authors.initials: M
authors.lastname: Egli
authors.affiliation: Alnylam Pharmaceuticals , 300 Third Street, Cambridge, Massachusetts 02142, United States.
authors.forename: Kallanthottathil G
authors.initials: KG
authors.lastname: Rajeev
authors.affiliation: Alnylam Pharmaceuticals , 300 Third Street, Cambridge, Massachusetts 02142, United States.
authors.forename: Muthiah
authors.initials: M
authors.lastname: Manoharan
mesh_terms: D001483:Base Sequence; D005095:Exoribonucleases / Q000737:chemistry*; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D009690:Nucleic Acid Conformation; D009841:Oligonucleotides / Q000737:chemistry*; D011743:Pyrimidines / Q000737:chemistry*; D013237:Stereoisomerism; D013816:Thermodynamics
chemical_list: D009841:Oligonucleotides; D011743:Pyrimidines; D005095:Exoribonucleases; C024843:poly A specific exoribonuclease