title: Stabilizing contributions of sulfur-modified nucleotides: crystal structure of a DNA duplex with 2'-O-[2-(methoxy)ethyl]-2-thiothymidines.
abstract: Substitution of oxygen atoms by sulfur at various locations in the nucleic acid framework has led to analogs such as the DNA phosphorothioates and 4'-thio RNA. The phosphorothioates are excellent mimics of DNA, exhibit increased resistance to nuclease degradation compared with the natural counterpart, and have been widely used as first-generation antisense nucleic acid analogs for applications in vitro and in vivo. The 4'-thio RNA analog exhibits significantly enhanced RNA affinity compared with RNA, and shows potential for incorporation into siRNAs. 2-Thiouridine (s2U) and 5-methyl-2-thiouridine (m5s2U) are natural nucleotide analogs. s2U in tRNA confers greater specificity of codon-anticodon interactions by discriminating more strongly between A and G compared with U. 2-Thio modification preorganizes the ribose and 2'-deoxyribose sugars for a C3'-endo conformation, and stabilizes heteroduplexes composed of modified DNA and complementary RNA. Combination of the 2-thio and sugar 2'-O-modifications has been demonstrated to boost both thermodynamic stability and nuclease resistance. Using the 2'-O-[2-(methoxy)ethyl]-2-thiothymidine (m5s2Umoe) analog, we have investigated the consequences of the replacement of the 2-oxygen by sulfur for base-pair geometry and duplex conformation. The crystal structure of the A-form DNA duplex with sequence GCGTAT*ACGC (T* = m5s2Umoe) was determined at high resolution and compared with the structure of the corresponding duplex with T* = m5Umoe. Notable changes as a result of the incorporation of sulfur concern the base-pair parameter 'opening', an improvement of stacking in the vicinity of modified nucleotides as measured by base overlap, and a van der Waals interaction between sulfur atoms from adjacent m5s2Umoe residues in the minor groove. The structural data indicate only minor adjustments in the water structure as a result of the presence of sulfur. The observed small structural perturbations combined with the favorable consequences for pairing stability and nuclease resistance (when combined with 2'-O-modification) render 2-thiouracil-modified RNA a promising candidate for applications in RNAi.
authors.affiliation: Department of Chemical Engineering, School of Engineering, Vanderbilt University, Nashville, TN 37235, USA.
authors.forename: Benjamin
authors.initials: B
authors.lastname: Diop-Frimpong
authors.forename: Thazha P
authors.initials: TP
authors.lastname: Prakash
authors.forename: Kallanthottathil G
authors.initials: KG
authors.lastname: Rajeev
authors.forename: Muthiah
authors.initials: M
authors.lastname: Manoharan
authors.forename: Martin
authors.initials: M
authors.lastname: Egli
mesh_terms: D020029:Base Pairing; D018360:Crystallography, X-Ray; D004247:DNA / Q000737:chemistry*; D008958:Models, Molecular; D009690:Nucleic Acid Conformation; D020320:Oligoribonucleotides, Antisense / Q000737:chemistry*; D013455:Sulfur / Q000737:chemistry; D013873:Thionucleotides / Q000737:chemistry*; D013936:Thymidine / Q000031:analogs & derivatives* / Q000737:chemistry*
chemical_list: D020320:Oligoribonucleotides, Antisense; D013873:Thionucleotides; D013455:Sulfur; D004247:DNA; D013936:Thymidine