2′‐Fluoro RNA Shows Increased Watson–Crick H‐Bonding Strength and Stacking Relative to RNA: Evidence from NMR and Thermodynamic Data

Patra, A.; Paolillo, Michael; Charissé, Klaus; Manoharan, Muthiah; Rozners, Eriks; Egli, Martin

doi:10.1002/anie.201204946

Cited by 76 publications

(67 citation statements)

References 35 publications

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“…One possibility is that an electron-donating 2′-amino substituent may affect the nucleobase p K a values or sugar puckering in a way that further weakens A:T base pairing, whereas a 3′-amino substituent, being further from the glycosidic linkage, might not have this effect. 33,34 Another example of a nucleobase-specific effect is our observation that an r(T) 4 template is significantly better than an r(U) 4 template in directing the polymerization of activated 3′-amino A monomer. Presumably this improvement reflects stronger or altered stacking interactions that cause the r(T) 4 template to be more ordered.…”

Section: Discussionmentioning

confidence: 97%

Synthesis of N3′-P5′-linked Phosphoramidate DNA by Nonenzymatic Template-Directed Primer Extension

Zhang

Blain

et al. 2013

J. Am. Chem. Soc.

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A fast and accurate pathway for nonenzymatic RNA replication would simplify models for the emergence of the RNA world from the prebiotic chemistry of the early earth. However, numerous difficulties stand in the way of an experimental demonstration of effective nonenzymatic RNA replication. To gain insight into the necessary properties of potentially self-replicating informational polymers, we have studied several model systems based on amino–sugar nucleotides. Here we describe the synthesis of N3′–P5′-linked phosphoramidate DNA (3′-NP-DNA) by the template-directed polymerization of activated 3′-amino-2′,3′-dideoxyribonucleotides. 3′-NP-DNA is an interesting model because of its very RNA-like A-type duplex conformation and because activated 3′-amino-2′,3′-dideoxyribonucleotides are much more reactive than the corresponding activated ribonucleotides. In contrast to our previous studies with 2′-amino-2′,3′-dideoxyribonucleotides (for which G and C but not A and T exhibit efficient template copying), we have found that all four canonical 3′-amino-2′,3′-dideoxyribonucleotides (G, C, A, and T) polymerize efficiently on RNA templates. RNA templates are generally superior to DNA templates, and oligo-ribo-T templates are superior to oligo-ribo-U templates, which are the least efficient of the RNA homopolymer templates. We have also found that activation of 3′-aminonucleotides with 2-methylimidazole results in a ca. 10-fold higher polymerization rate relative to activation with imidazole, an observation that parallels earlier findings with ribonucleotides. We discuss the implications of our experiments for the possibility of self-replication in the 3′-NP-DNA and RNA systems.

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Section: Discussionmentioning

confidence: 97%

Synthesis of N3′-P5′-linked Phosphoramidate DNA by Nonenzymatic Template-Directed Primer Extension

Zhang

Blain

et al. 2013

J. Am. Chem. Soc.

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“…The 2 -deoxy-2 -fluororibonucleotide (2 -F-RNA) modification has been widely used to modify oligonucleotides for oligonucleotidebased therapeutics. Incorporation of a 2 -F-RNA residue stabilizes an A-form RNA duplex (Egli & Manoharan, 2019;Manoharan et al, 2011;Patra et al, 2012). Specifically, 2 -F ribo-substitution pre-organizes the sugar into a C3 -endo or North conformation and hence reduces the entropic penalty for the formation of the A-form duplex and increases base stacking and Watson-Crick hydrogen-bond stabilities due to its electronwithdrawing power (Patra et al, 2012).…”

Section: Background Informationmentioning

confidence: 99%

“…Incorporation of a 2 -F-RNA residue stabilizes an A-form RNA duplex (Egli & Manoharan, 2019;Manoharan et al, 2011;Patra et al, 2012). Specifically, 2 -F ribo-substitution pre-organizes the sugar into a C3 -endo or North conformation and hence reduces the entropic penalty for the formation of the A-form duplex and increases base stacking and Watson-Crick hydrogen-bond stabilities due to its electronwithdrawing power (Patra et al, 2012). Furthermore, 2 -F-RNA-modified siRNAs have reduced immune stimulation and improved activity in vitro and in vivo compared to unmodified siRNAs and are in clinical development.…”

Section: Background Informationmentioning

confidence: 99%

Synthesis of 2′‐Fluorinated Northern Methanocarbacyclic (2′‐F‐NMC) Nucleosides and Their Incorporation Into Oligonucleotides

Akabane‐Nakata

Kumar

Erande

et al. 2020

CP Nucleic Acid Chemistry

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This article describes chemical synthesis of 2′‐fluorinated Northern methanocarbacyclic (2′‐F‐NMC) nucleosides and phosphoramidites, based on a bicyclo[3.1.0]hexane scaffold bearing all four natural nucleobases (U, C, A, and G), and their incorporation into oligonucleotides by solid‐supported synthesis. This synthesis starts from commercially available cyclopent‐2‐en‐1‐one to obtain the fluorinated carbocyclic pseudosugar intermediate (S.13), which can be converted to the uridine intermediate by condensation with isocyanate, followed by cyclization, and to adenine and guanine precursors by microwave‐assisted reactions. All four 2′‐F‐NMC phosphoramidites are synthesized from S.13 in a convergent approach, and the monomers are used for synthesis of 2′‐F‐NMC‐modified oligonucleotides. © 2020 by John Wiley & Sons, Inc. Basic Protocol 1: Preparation of fluorinated carbocyclic pseudosugar intermediate Basic Protocol 2: Preparation of 2′‐F‐NMC uridine and cytidine phosphoramidites Basic Protocol 3: Preparation of 2′‐F‐NMC adenosine phosphoramidite Basic Protocol 4: Preparation of 2′‐F‐NMC guanosine phosphoramidite Basic Protocol 5: Synthesis of oligonucleotides containing 2ʹ‐F‐NMC

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“…We also incorporated 2’-F-RNAs in this round of optimization since they can increase thermal and nuclease stability of RNA:RNA or RNA:DNA duplexes, and they also interfere minimally with C3’-endo sugar puckering. 21,22 2’-F may be better tolerated than 2’-OMe at positions where the 2’-OH is important for RNA:DNA duplex stability. For these reasons, we synthesized two crRNAs based on C9 but with 2’-F modifications at positions 11-14 and/or 17-18 ( C10-C11 ).…”

Section: Figurementioning

confidence: 99%

Heavily and Fully Modified RNAs Guide Efficient SpyCas9-Mediated Genome Editing

Mir

Alterman

Hassler

et al. 2018

Preprint

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Synthesis of Oligonucleotides. CRISPR guides were synthesized at 1 µmole scale on an Applied Biosystems 12 394 DNA synthesizer. BTT (0.25 M in acetonitrile, ChemGenes) was used as activator. 0.05 M iodine in 13 pyridine:water (9:1) (TEDIA) was used as oxidizer. DDTT (0.1 M, ChemGenes) was used as sulfurizing 14 agent. 3% TCA in DCM (TEDIA) was used as deblock solution. Oligonucleotides were grown on 1000 Å 15 CPG functionalized with Unylinker (~42 µmol/g). RNA and 2'-OMe phosphoramidites (ChemGenes) were 16 dissolved in acetonitrile to 0.15 M; the coupling time was 10 min for each base. The nucleobases were 17 deprotected with a 3:1 NH4OH:EtOH solution for 48 hours at room temperature. Deprotection of the 18 TBDMS group was achieved with DMSO:NEt3•3HF (4:1) solution (500 µL) at 65 °C for 3 hours. RNA 19oligonucleotides were then recovered by precipitation in 3M NaOAc (25 µL) and n-BuOH (1 mL), and the 20 pellet was washed with cold 70% EtOH and resuspended in 1 mL RNase-free water. 21

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2′‐Fluoro RNA Shows Increased Watson–Crick H‐Bonding Strength and Stacking Relative to RNA: Evidence from NMR and Thermodynamic Data

Cited by 76 publications

References 35 publications

Synthesis of N3′-P5′-linked Phosphoramidate DNA by Nonenzymatic Template-Directed Primer Extension

Synthesis of N3′-P5′-linked Phosphoramidate DNA by Nonenzymatic Template-Directed Primer Extension

Synthesis of 2′‐Fluorinated Northern Methanocarbacyclic (2′‐F‐NMC) Nucleosides and Their Incorporation Into Oligonucleotides

Heavily and Fully Modified RNAs Guide Efficient SpyCas9-Mediated Genome Editing

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