Large-Scale Solid-Phase Preparation of 3′-Unprotected Trinucleotide Phosphotriesters - Precursors for Synthesis of Trinucleotide Phosphoramidites

Kayushin, A.; Korosteleva, Maria; Мирошников, А. И.

doi:10.1080/15257770008045471

Cited by 17 publications

(19 citation statements)

References 7 publications

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“…Here it is of utmost importance that cleavage of the trinucleotide from the support proceeds under conditions that leave all other protecting groups intact. Controlled pore glass (CPG) with the start nucleoside linked via an oxalyl anchor has been used in this regard and was found to be suitable for large scale production of trinucleotides (Kayushin, Korosteleva, & Miroshnikov, 2000). Recently, soluble supports were recognized as suitable alternatives to solid supports in trinucleotide synthesis (Kungurtsev et al, 2016).…”

Section: Background Informationmentioning

confidence: 99%

Synthesis of Trinucleotide Building Blocks in Solution and on Solid Phase

Suchsland

Appel

Müller

2018

CP Nucleic Acid Chemistry

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We have developed two methods, in solution and on solid phase, that give easy access to trinucleotide phosphoramidites capable of undergoing coupling reactions by the solid‐phase phosphoramidite approach. The solution protocol is characterized by application of 5′‐O‐dimethoxytrityl (DMT) and 3′‐O‐tert‐butyldimethylsilyl (TBDMS) as a pair of orthogonal protecting groups and 2‐cyanoethyl (CE) for protection of the phosphate. Starting with suitably functionalized monomers, synthesis proceeds in the 3′‐ to 5′‐direction, delivering the fully protected trinucleotide. The 3′‐O‐protecting group is cleaved followed by phosphitylation of the free 3′‐OH group. The solid‐phase protocol is based on standard phosphoramidite chemistry in conjunction with a dithiomethyl linkage connecting the 3′‐starting nucleoside to the polymer. The disulfide bridge can be cleaved under neutral conditions for release of the trinucleotide from the support preserving all other protecting groups. © 2018 by John Wiley & Sons, Inc.

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Section: Background Informationmentioning

confidence: 99%

Synthesis of Trinucleotide Building Blocks in Solution and on Solid Phase

Suchsland

Appel

Müller

2018

CP Nucleic Acid Chemistry

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“…Primers encompassing the desired sequence diversity were synthesized, with high diversity regions constructed using a mixed pool of trimer phosphoramidites to match the target codon frequency 9,10 . We then built a pooled DNA library encoding the full nanobody sequence by assembly PCR of these primers (see Methods for full details).…”

Section: Library Construction and Validationmentioning

confidence: 99%

Platform for rapid nanobody discoveryin vitro

McMahon

Zheng

et al. 2017

Preprint

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Camelid single-domain antibody fragments ("nanobodies") provide the remarkable specificity of antibodies within a single immunoglobulin V HH domain. This unique feature enables applications ranging from their use as biochemical tools to therapeutic agents. Virtually all nanobodies reported to date have been obtained by animal immunization, a bottleneck restricting many applications of this technology. To solve this problem, we developed a fully in vitro platform for nanobody discovery based on yeast surface display of a synthetic nanobody scaffold. This platform provides a facile and cost-effective method for rapidly isolating nanobodies targeting a diverse range of antigens. We provide a blueprint for identifying nanobodies starting from both purified and non-purified antigens, and in addition, we demonstrate application of the platform to discover rare conformationally-selective nanobodies to a lipid flippase and a G protein-coupled receptor. To facilitate broad deployment of this platform, we have made the library and all associated protocols publicly available. IntroductionAntibodies have had a transformative impact on science and medicine due to their exceptional specificity and biochemical versatility, enabling applications in almost every aspect of biomedical inquiry. Conventional antibodies are composed of two heavy chains and two light chains. Each chain contributes to antigen binding specificity through a variable domain, termed V H and V L for the heavy and light chain, respectively. A key exception to this general architecture is found in camelids (llamas, alpacas, and their relatives), which possess a parallel antibody repertoire composed solely of heavy chains 1,2 . Such antibodies bind to their target antigens through a single variable domain, termed V HH , which contains the entire antigen-binding surface. Unlike the antigen binding fragments of conventional antibodies (Fabs), isolated V HH domains (also called "nanobodies") can be readily expressed in bacteria as the product of a single gene, and in many cases these fragments can even fold and retain antigen specificity in the reducing environment of the cytosol. Owing to their versatility, nanobodies have found applications in protein biochemistry and structural biology, cell biology, and as potential diagnostic and therapeutic agents [2][3][4][5][6] .

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“…Furthermore, to obtain better control of the level of diversification, a combined synthesis approach using Fmoc‐protected trinucleotides together with DMTr‐protected mononucleotides has been devised . However, trinucleotide phosphoramidities are more difficult and expensive to produce, although progress has been made for large‐scale solid phase synthesis . The Slonomics™ technique is another elegant approach to produce non‐biased randomized oligonucleotides, exploiting the advantage of triplet‐based building blocks, while avoiding any requirement of presynthesized trinucleotides.…”

Section: Generation Of Library Diversitymentioning

confidence: 99%

“…78 However, trinucleotide phosphoramidities are more difficult and expensive to produce, although progress has been made for large-scale solid phase synthesis. 79 The Slonomics technique 80 is another elegant approach to produce non-biased randomized oligonucleotides, exploiting the advantage of triplet-based building blocks, while avoiding any requirement of presynthesized trinucleotides. In this technique, universal building blocks are synthesized as single-stranded oligonucleotides forming double-stranded hairpin structures having trinucleotide single-stranded overhangs.…”

Section: Oligonucleotide-directed Randomizationmentioning

confidence: 99%

Affinity proteins and their generation

Ståhl

Kronqvist

Jonsson

et al. 2012

J of Chemical Tech & Biotech

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Engineered affinity proteins have, together with antibodies and antibody derivatives, become indispensable tools in many areas of life science and with an increasing number of applications. The need for high‐throughput methods for generation of these different affinity proteins is evident. Today, combinatorial protein engineering is the most successful strategy to generate novel affinity proteins of non‐immunoglobulin origin. In this approach, high‐complexity combinatorial libraries are constructed from which affinity proteins are isolated using appropriate selection methods, thus circumventing the need for detailed knowledge of the protein structure and the binding mechanism that is necessary in more rational approaches. Since the introduction of the phage display technology, several alternative selection systems have been developed for this purpose. This review presents briefly some of the more commonly used affinity proteins, and gives an overview of the different methods and challenges related to the generation of library diversity and the selection methods available for the isolation of affinity proteins with desired properties. Copyright © 2012 Society of Chemical Industry

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Large-Scale Solid-Phase Preparation of 3′-Unprotected Trinucleotide Phosphotriesters - Precursors for Synthesis of Trinucleotide Phosphoramidites

Cited by 17 publications

References 7 publications

Synthesis of Trinucleotide Building Blocks in Solution and on Solid Phase

Synthesis of Trinucleotide Building Blocks in Solution and on Solid Phase

Platform for rapid nanobody discoveryin vitro

Affinity proteins and their generation

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