Protocol for the Solid-phase Synthesis of Oligomers of RNA Containing a 2'-<em>O</em>-thiophenylmethyl Modification and Characterization via Circular Dichroism

Abstract

Solid-phase synthesis has been used to obtain canonical and modified polymers of nucleic acids, specifically of DNA or RNA, which has made it a popular methodology for applications in various fields and for different research purposes. The procedure described herein focuses on the synthesis, purification, and characterization of dodecamers of RNA 5'-[CUA CGG AAU CAU]-3' containing zero, one, or two modifications located at the C2'-O-position. The probes are based on 2-thiophenylmethyl groups, incorporated into RNA nucleotides via standard organic synthesis and introduced into the corresponding oligonucleotides via their respective phosphoramidites. This report makes use of phosphoramidite chemistry via the four canonical nucleobases (Uridine (U), Cytosine (C), Guanosine (G), Adenosine (A)), as well as 2-thiophenylmethyl functionalized nucleotides modified at the 2'-O-position; however, the methodology is amenable for a large variety of modifications that have been developed over the years. The oligonucleotides were synthesized on a controlled-pore glass (CPG) support followed by cleavage from the resin and deprotection under standard conditions, i.e., a mixture of ammonia and methylamine (AMA) followed by hydrogen fluoride/triethylamine/N-methylpyrrolidinone. The corresponding oligonucleotides were purified via polyacrylamide electrophoresis (20% denaturing) followed by elution, desalting, and isolation via reversed-phase chromatography (Sep-pak, C18-column). Quantification and structural parameters were assessed via ultraviolet-visible (UV-vis) and circular dichroism (CD) photometric analysis, respectively. This report aims to serve as a resource and guide for beginner and expert researchers interested in embarking in this field. It is expected to serve as a work-in-progress as new technologies and methodologies are developed. The description of the methodologies and techniques within this document correspond to a DNA/RNA synthesizer (refurbished and purchased in 2013) that uses phosphoramidite chemistry.

Folding Coarse-Grained Oligomer Models with PyRosetta

“…24 These solid-phase synthesis methods suffer from poor protective reaction conditions resulting in low polymer length and low final product yield. 25,26 Furthermore, methods for foldamer characterization have additional challenges compared to more well-established biopolymer chacterization due to unknown spectroscopic signatures of new foldamer molecules. 1 In addition, the design of novel backbone chemistry that folds in a desired solvent is some what of an art.…”

“…1,2 Biosimilar foldamers also take advantage of preexisting synthesis techniques allowing comparatively quick synthesis compared to a novel backbone synthesis. 24,27,28 Despite their similarity to existing biopolymers, these biosimilar foldamers are still considerably difficult to design. 1 In order to facilitate the foldamer design process, new ways to propose potentially stable foldamers chemistries are needed.…”

Folding Coarse-Grained Oligomer Models with PyRosetta

“…Most foldamers that resemble existing biopolymers are synthesized using solid-phase synthesis for their respective linker bond type . These solid-phase synthesis methods suffer from poor protective reaction conditions resulting in low polymer length and low final product yield. , Furthermore, methods for foldamer characterization have additional challenges compared to more well-established biopolymer characterization due to unknown spectroscopic signatures of new foldamer molecules …”

“…Current success in foldamer design has stemmed from several biosimilar foldamer backbones that borrow many design choices from traditional biopolymers. , Using peptide or phosphodiester linkers allows researchers to start with a working design that has been evolutionarily selected to fold under certain conditions. These biopolymers include interactions such as hydrogen bonds in protein backbones, or π-stacking and base-pair hydrogen bonds in nucleic acids. , Biosimilar foldamers also take advantage of pre-existing synthesis techniques allowing comparatively quick synthesis compared to a novel backbone synthesis. ,, Despite their similarity to existing biopolymers, these biosimilar foldamers are still considerably difficult to design . In order to facilitate the foldamer design process, new ways to propose potentially stable foldamer chemistries are needed.…”

“…23 protective reaction conditions resulting in low polymer length and low final product yield. 24,25 Furthermore, methods for foldamer characterization have additional challenges compared to more well-established biopolymer characterization due to unknown spectroscopic signatures of new foldamer molecules. 1 In addition, the design of novel backbone chemistry that folds in a desired solvent is somewhat of an art.…”