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Commercial Scale Manufacturing of Oligonucleotides Under Good Manufacturing Practices
Published in Eric Wickstrom, Clinical Trials of Genetic Therapy with Antisense DNA and DNA Vectors, 2020
Jose E. Gonzalez, Richard G. Einig, Patricia Puma, Timothy P. Noonan, Paul E. Kennedy, Bruce G. Sturgeon, Bing H. Wang, Jin-yan Tang
The use of dimer block synthons halves the oligonucleotide synthesis cycle time, and reduces the use of solvents and other reagents, while doubling the oligonucleotide plant synthetic throughput capacity, and increasing crude quality and yield. The key advantages of solution phase production are the possibility of very large scale operation and high efficiency in monomer use, and relatively low solvent consumption. However, its drawback is that it cannot maintain a high level of purity for oligonucleotides more than 8 bases long. In contrast, solid phase synthesis excels at making high purity, longer oligonucleotides.
In situ Hybridization Histochemistry
Published in Edythe D. London, Imaging Drug Action in the Brain, 2017
Martin K.-H. Schofer, James P. Herman, Stanley J. Watson
Several characteristics of oligonucleotide probes render them quite attractive for in situ experiments. First, they are by far the easiest of three types to acquire, as they can be generated to any published nucleotide sequence. Most molecular biology departments have an automatic oligonucleotide synthesizer available; in addition, many commercial sources offer custom oligonucleotide synthesis. Second, oligonucleotides are quite easy to use; generally, in situ hybridization using oligonucleotides does not require extensive molecular biological skill or experience. Third, probe composition and length can easily be designed on paper by the investigator, allowing for analysis of specific regions of the mRNA in question. However, it is required that probe design be very carefully planned to ensure specificity for the particular RNA of interest (Agarwal et al., 1981). For example, one does not want to use oligonucleotides directed against sequences common to many DNA or RNA molecules, such as DNA binding domains of steroid receptors and related compounds (Evans, 1988) or highly conserved transmembrane spanning domains of membrane-bound receptors (Bunzow et al., 1988).
Protein Subunit Vaccines and Recombinant DNA Technology
Published in F. Y. Liew, Vaccination Strategies of Tropical Diseases, 2017
With the recent introduction of fully automated oligonucleotide-synthesis machines, the generation of oligomers up to and over 100 bases is now possible.25 Thus, if the protein sequence is known, DNA encoding these proteins can be synthesized. A series of overlapping fragments are generated and are sequentially ligated together, the final result being checked by DNA sequencing, as single base change mutants can arise. The advantage of this approach is that the whole system can be designed at once, such that as soon as the DNA sequence has been generated it can be inserted into a plasmid and expressed, all the correct signals for expression having been engineered already.
Advances in biocatalytic and chemoenzymatic synthesis of nucleoside analogues
Published in Expert Opinion on Drug Discovery, 2022
Sebastian C. Cosgrove, Gavin J. Miller
It is sensible to consider this prospect of using enzymes to perform classical synthetic nucleoside-producing reactions in a broader context. For example, complex glycan synthesis has benefitted immeasurably from the development of glycosyltransferases to assemble (in part or in full) complex oligosaccharide targets for biological and biomedical applications [47–51]. In addition, and comparatively to the example reviewed earlier for the provision of ribose 1-phosphates, the provision of the key sugar nucleotide building blocks to achieve this has also flourished [52–56]. Similarly, looking to classical oligonucleotide synthesis [57], the phosphoramidite method of assembling these essential sequences has been challenged recently not only by new chemical approaches [58–60] but also by using enzyme platforms to achieve biopolymer synthesis [61,62]. Considering the enabling effect of introducing biocatalytic approaches into these fields, the future prospects for nucleoside analogue chemistry translate to one of significant potential.
A comprehensive search of functional sequence space using large mammalian display libraries created by gene editing
Published in mAbs, 2019
Kothai Parthiban, Rajika L. Perera, Maheen Sattar, Yanchao Huang, Sophie Mayle, Edward Masters, Daniel Griffiths, Sachin Surade, Rachael Leah, Michael R. Dyson, John McCafferty
We illustrate the potential of this approach by chain shuffling a population of variable heavy (VH) genes derived by single-chain variable fragment (scFv) phage display, reformatting into IgG-format, and creating and screening a mammalian display library. In addition, we have affinity matured a programmed cell death protein 1 (PD-1)-blocking antibody by oligonucleotide-directed mutagenesis. Random mutagenesis has routinely been used to create variant libraries from starting clones in search of improved variants. The availability of modern high throughput oligonucleotide synthesis technology, however, means that a more directed approach can be taken to library design and construction. By this approach, it becomes possible to retain much of the core sequence while still sampling variants around that sequence space. The potential of combining this approach with mammalian display was exemplified by using CRISPR/Cas9 or TALE nucleases, to create diversified libraries in an IgG format. These were subjected to stringent selection to identify critical residues involved in the interaction, resulting in higher affinity blocking antibodies.
Protective effect of Gelofusine against cRGD-siRNA-induced nephrotoxicity in mice
Published in Renal Failure, 2018
Wenjie Liao, Yixin Qin, Lumin Liao, Bohong Cen, Zhuomin Wu, Yuanyi Wei, Zhen Wang, Guoxian Li, Aimin Ji
The siRNA sequence employed for our experiments was human VEGFR2 siRNA. The sense strand was 5′-mGmAmGAACCUCACAUGGUmAmCmA dTdT-3′ and the antisense strand was 5′-mUmGmUACCAUGUGAGGU-UmCmUmC dTdT-3′. Cy5 phosphoramidites (RiboBio Co., Ltd., Guangzhou, China) were attached to the 5′-ends of the siRNA antisense strands. The synthesis and purification of the sense strand and the antisense strand were based on standard oligonucleotide synthesis and deprotection protocols [19]. Cyclo (Arg-Gly-Asp-D-Phe-Lys[PEG-MAL]) (cRGD) peptides, where PEG is 8-amino-3,6-dioxaoctanoic acid and MAL is β-maleimidopropionic acid, were synthesized by Peptide International (Louisville, KY). The conjugation of cRGD-siRNA was performed as follows. The 5′-thiol-modified siRNA sense strand was dissolved in HEPES-KOH buffer (pH 7.2) and cRGD was then added. N2 was used to saturate the solution to remove air and the Michael reaction was performed at 4 °C. The resulting solution was filtered via centrifugation (MW = 3000) and then washed three times with RNase-free water to remove excess cRGD.