Chemical Synthesis of Modified RNA

Höbartner, Claudia; Wachowius, Falk

doi:10.1002/9780470664001.ch1

Cited by 12 publications

(8 citation statements)

References 145 publications

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“…In addition to X-ray crystallography and NMR techniques, improvements in solid-phase synthesis of modified oligonucleotides (Verma and Eckstein, 1998 ; Höbartner and Wachowius, 2010 ), together with the use of fluorescent nucleotide analogs (Jameson and Eccleston, 1997 ; Cremo, 2003 ) and more efficient post-synthetic labeling strategies (Rublack et al, 2011 ), has allowed a remarkable increase in the application of fluorescence-based methods to characterize aptamer-ligand interactions. In particular, the emergence of fluorescence microscopy techniques that allow monitoring the dynamics of individual structures has revolutionized our understanding of nucleic acid function and structure (McCluskey et al, 2014 ; Boudreault et al, 2015 ; Perez-Gonzalez and Penedo, 2015 ).…”

Section: Biophysical Methods To Investigate Aptamer-ligand Complexesmentioning

confidence: 99%

Fluorescence-Based Strategies to Investigate the Structure and Dynamics of Aptamer-Ligand Complexes

2016

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In addition to the helical nature of double-stranded DNA and RNA, single-stranded oligonucleotides can arrange themselves into tridimensional structures containing loops, bulges, internal hairpins and many other motifs. This ability has been used for more than two decades to generate oligonucleotide sequences, so-called aptamers, that can recognize certain metabolites with high affinity and specificity. More recently, this library of artificially-generated nucleic acid aptamers has been expanded by the discovery that naturally occurring RNA sequences control bacterial gene expression in response to cellular concentration of a given metabolite. The application of fluorescence methods has been pivotal to characterize in detail the structure and dynamics of these aptamer-ligand complexes in solution. This is mostly due to the intrinsic high sensitivity of fluorescence methods and also to significant improvements in solid-phase synthesis, post-synthetic labeling strategies and optical instrumentation that took place during the last decade. In this work, we provide an overview of the most widely employed fluorescence methods to investigate aptamer structure and function by describing the use of aptamers labeled with a single dye in fluorescence quenching and anisotropy assays. The use of 2-aminopurine as a fluorescent analog of adenine to monitor local changes in structure and fluorescence resonance energy transfer (FRET) to follow long-range conformational changes is also covered in detail. The last part of the review is dedicated to the application of fluorescence techniques based on single-molecule microscopy, a technique that has revolutionized our understanding of nucleic acid structure and dynamics. We finally describe the advantages of monitoring ligand-binding and conformational changes, one molecule at a time, to decipher the complexity of regulatory aptamers and summarize the emerging folding and ligand-binding models arising from the application of these single-molecule FRET microscopy techniques.

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Section: Biophysical Methods To Investigate Aptamer-ligand Complexesmentioning

confidence: 99%

Fluorescence-Based Strategies to Investigate the Structure and Dynamics of Aptamer-Ligand Complexes

2016

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“…For successful solid-phase RNA synthesis, the phosporamidite monomeric units must be protected with a combination of orthogonal R 1 transient and R 2 , R 3 , R 4 permanent protecting groups. Several synthetic protocols, systems of protecting groups, and deprotection strategies have been demonstrated so far [ 38 ]. In practice, the building blocks are protected by employing one of the five most common strategies: (1) 5’- O -DMTr-2’- O -TBDMS [ 39 , 40 , 41 ]; (2) 5’- O -DMTr-2’- O -TOM [ 42 ]; (3) 5’- O -DMTr-2’- O -Fpmp [ 43 ]; (4) 5’- O -DMTr-2’- O -TC [ 44 ], and (5) 5’- O -DOD(BzH)-2’- O -ACE [ 45 , 46 ] ( Figure 2 ).…”

Section: Solid-phase Synthesis Of Modified Rna Oligomers Via Phospmentioning

confidence: 99%

Synthesis of Nucleobase-Modified RNA Oligonucleotides by Post-Synthetic Approach

et al. 2020

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The chemical synthesis of modified oligoribonucleotides represents a powerful approach to study the structure, stability, and biological activity of RNAs. Selected RNA modifications have been proven to enhance the drug-like properties of RNA oligomers providing the oligonucleotide-based therapeutic agents in the antisense and siRNA technologies. The important sites of RNA modification/functionalization are the nucleobase residues. Standard phosphoramidite RNA chemistry allows the site-specific incorporation of a large number of functional groups to the nucleobase structure if the building blocks are synthetically obtainable and stable under the conditions of oligonucleotide chemistry and work-up. Otherwise, the chemically modified RNAs are produced by post-synthetic oligoribonucleotide functionalization. This review highlights the post-synthetic RNA modification approach as a convenient and valuable method to introduce a wide variety of nucleobase modifications, including recently discovered native hypermodified functional groups, fluorescent dyes, photoreactive groups, disulfide crosslinks, and nitroxide spin labels.

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“…The latest advances in chemical RNA synthesis have recently been reviewed [1][2][3]. The four steps of the synthesis cycle include: (A) cleavage of the transient 5 0 -protecting group; (B) activation of the phosphoramidite building block and coupling to the 5 0 -OH of the support-bound nucleotide; (C) capping of unreacted 5 0 -termini to prevent subsequent extension; and (D) oxidation of the phosphite triester to a phosphate triester internucleotide bond.…”

Section: Rna Solid-phase Synthesismentioning

confidence: 99%

“…The latest advances in chemical RNA synthesis have recently been reviewed [1][2][3]. Synthetic RNA oligonucleotides are also indispensable tools for structural studies and the biochemical analyses of RNA-RNA or RNA-protein interactions.…”

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confidence: 99%

Chemical Synthesis of RNA

Höbartner

2012

Alternative Pre‐mRNA Splicing

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The discovery of RNA interference and the therapeutic potential for modified RNA underline the growing importance of synthetic RNA in basic biomedical research. Synthetic RNA oligonucleotides are also indispensable tools for structural studies and the biochemical analyses of RNA-RNA or RNA-protein interactions. The chemical synthesis of RNA offers the unique possibility to introduce site-specific modifications and attachment sites for biophysical labels. In this chapter, chemical modification strategies for RNA are summarized, and a brief overview is provided of the incorporation of modified oligonucleotides into larger RNA constructs by enzymatic ligation.14.1 Theoretical Background RNA Solid-Phase SynthesisThe automated chemical synthesis of RNA oligonucleotides consists of the repeated coupling of ribonucleoside phosphoramidite building blocks on a solid support. The four steps of the synthesis cycle include: (A) cleavage of the transient 5 0 -protecting group; (B) activation of the phosphoramidite building block and coupling to the 5 0 -OH of the support-bound nucleotide; (C) capping of unreacted 5 0 -termini to prevent subsequent extension; and (D) oxidation of the phosphite triester to a phosphate triester internucleotide bond. The four steps are repeated until the desired oligonucleotide length is assembled. The full-length RNA is then released from the solid support to which it was attached via its 3 0 -hydroxyl group. At the same time, nucleobase and phosphate-protecting groups are removed. Cleavage of the 2 0 -protecting groups affords then the final oligoribonucleotide product. In the vast majority of chemistries, and in all commercially available chemistries, the 3 0 nucleotide is linked to the column. This phosphoramidite-based RNA solid-phase synthesis cycle is highly similar to standard automated DNA solid-phase synthesis, but the requirement for additional 2 0 -protecting groups makes RNA synthesis much more challenging. The key to successful solid-phase RNA synthesis is the choice of a suitable combination of orthogonal protecting groups (R, R 1 , R 2 , R 3 ). It is of critical importance that the 2 0 -protecting groups (R 2 ) remain completely intact until the final deprotection step, and that they can be removed under conditions that do not affect the integrity of the target RNA. The increasing demand for synthetic RNA oligonucleotides has spurred renewed efforts in the development of new protecting group strategies, with the goal to render RNA synthesis as efficient and reliable as DNA synthesis. The latest advances in chemical RNA synthesis have recently been reviewed [1][2][3]. Presently, the three most important families of phosphoramidite Alternative pre-mRNA Splicing: Theory and Protocols, First Edition. Edited

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Chemical Synthesis of Modified RNA

Cited by 12 publications

References 145 publications

Fluorescence-Based Strategies to Investigate the Structure and Dynamics of Aptamer-Ligand Complexes

Fluorescence-Based Strategies to Investigate the Structure and Dynamics of Aptamer-Ligand Complexes

Synthesis of Nucleobase-Modified RNA Oligonucleotides by Post-Synthetic Approach

Chemical Synthesis of RNA

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