RNA and Viruses Biotechnology Lecture 6 PDF

Lecture 6 RNA and viruses and Biotechnology MMB-505 Principles of RNA biology Jenni Pedor  Overview Viruses Enzymes Viral IRES Viral tRNAs tRNA in biotech mRNA in biotech miRNA/siRNA in biotech What to p...

Lecture 6 RNA and viruses and Biotechnology MMB-505 Principles of RNA biology Jenni Pedor  Overview Viruses Enzymes Viral IRES Viral tRNAs tRNA in biotech mRNA in biotech miRNA/siRNA in biotech What to prepare for the last seminar What to expect on the exam 07/10/2024 2  Next gen RTs in tRNA-seq Challenges in tRNA-seq: secondary structures nucleotide modifications Next generation reverse transcriptases Read through secondary structures Read through modifications Can be used for Quantitative analysis of expressed tRNAs tRNA-seq Ribosome profiling … 07/10/2024 3  The Baltimore classification https://viralzone.expasy.org/254 07/10/2024 4  RTs of retroelements - elements that are transcribed into RNA, reverse-transcribed into DNA and then inserted into a new site in the genome. Infectious retroviruses Other retroelements Retroviral RTs -genetic parasites that inhabit the genomes of all eukaryotes and many prokaryotes Maxima RT (MMLV, ThermoFisher Scientific) SuperScript IV (MMLV, ThermoFisher Scientific) ProtoScript II (MMLV, New England Biolabs) Mobile group II introns (retrotransposons) Next generation RTs TGIRT (Ingex) MarathonRT (RNAConnect) Induro (NEB) https://www.ncbi.nlm.nih.gov/mesh?Db=mesh&Cmd=DetailsSearch&Term=%22Retroelements%22%5BMeSH+Terms%5D 07/10/2024 5 Boeke JD, Stoye JP. Retrotransposons, Endogenous Retroviruses, and the Evolution of Retroelements. In: Coffin JM, Hughes SH, Varmus HE, editors. Retroviruses. 07/10/2024 6 Cold Spring Harbor (NY): Cold Spring Harbor Laboratory Press; 1997. Available from: https://www.ncbi.nlm.nih.gov/books/NBK19468/  Mobile group II introns High processivity and fidelity (naturally synthetize >2-kb transcripts) First purified Lactococcus lactis Ll.LtrB intron LtrA (Saldanha et al. 1999) Low error rate ∼10^−5 (Conlan et al. 2005) Difficult to produce (Vellore et al. 2004; Chee and Takami 2005; Ng et al. 2007). Poorly expressed Insoluble in absence of bound RNA TGIRT - Thermostable group II intron encoded RT from Geobacillus stearothermophilus (Mohr et al. 2013) MarathonRT - group II intron maturase from Eubacterium rectale (Zhao and Pyle 2016) 07/10/2024 7  TGIRT first description (Mohr et al., 2013) TGIRT TGIRT TGIRT RT activity at elevated temperatures, high processivity TGIRT Less premature stops (highly structured stable intron RNA as a template) Lower error rate 07/10/2024 8 Mohr, S. et al. Thermostable group II intron reverse transcriptase fusion proteins and their use in cDNA synthesis and next-generation RNA sequencing. RNA 19, 958–970 (2013). Mim-tRNAseq (Behrens et al. 2021) 07/10/2024 9 Behrens, A. et al. High-resolution quantitative profiling of tRNA abundance and modification status in eukaryotes by mim-tRNAseq. Mol. Cell 1–14 (2021) doi:10.1016/j.molcel.2021.01.028.  Template switching Primer duplex RNA template RNA 5’ 3’ 3’ 5’ DNA Certain RTs can jump from one template strand to another RNA strand that is in the immediate neighborhood, where they pick up and resume cDNA synthesis Discovered in the 1970-1990s during studies of retroviral replication (Coffin 1979, Gilboa et al. 1979, Peliska and Benkovic 1992) Mechanism patented for the synthesis of cDNA by Alan Lambowitz 07/10/2024 10 TGIRT bias during template switching (Xu et al. 2019) Uncorrected proof PDF available only DNA/RNA ligase TGIRT template switching Xu, H., Yao, J., Wu, D. C. & Lambowitz, A. M. Improved TGIRT-seq methods for comprehensive transcriptome profiling with decreased adapter dimer formation and bias correction. Sci. Rep. 9, 7953 (2019). Template switch bias can be remediated computationally. 07/10/2024 11  TGIRT Has not been commercially available for few years New InGex website with Chinese domain http://www.ingex.cn/a/chanpinzhongxin/mei/2021/0811/21.html 07/10/2024 12 MRT activity and mutants (Zhao et al., 2018) 07/10/2024 13 Zhao, C., Liu, F. & Pyle, A. M. An ultraprocessive, accurate reverse transcriptase encoded by a metazoan group II intron. RNA 24, 183–185 (2018).  MarathonRT Sold by RNAConnect – a company set up to sell uMRT RT enzyme, available since 2024 https://rnaconnect.com/ 07/10/2024 14  Mobile group II intron RTs https://www.neb.com/en/products/m0681-induro-reverse-transcriptase#Product%20Information 07/10/2024 15  Enzymes from bacteriophages T4 RNA ligase 2, truncated T4 Polynucleotide Kinase T7 polymerase CircLigase from TS2126 Yap ML, Rossmann MG. Structure and function of bacteriophage T4. Future Microbiol. 2014;9(12):1319-27. doi: 10.2217/fmb.14.91. PMID: 25517898; PMCID: PMC4275845. 07/10/2024 16  T4 PNK T4 Polynucleotide Kinase from Bacteriophage T4 T4 Polynucleotide Kinase (3' phosphatase minus) T4 Polynucleotide Kinase 5' phosphorylation of DNA/RNA for subsequent ligation End labeling DNA or RNA for probes and DNA sequencing Removal of 3' phosphoryl groups Catalyzes the transfer and exchange of Pi from the γ position of ATP to the 5´ -hydroxyl terminus of polynucleotides (double-and single-stranded DNA and RNA) and nucleoside 3´-monophosphates. Polynucleotide Kinase also catalyzes the removal of 3´-phosphoryl groups from 3´-phosphoryl polynucleotides, deoxynucleoside 3´-monophosphates and deoxynucleoside 3´-diphosphates. https://www.neb.com/en/products/m0201-t4-polynucleotide-kinase#Product%20Information 07/10/2024 17  T4 DNA/RNA ligase Bacteriophage T4 RNA Ligase 2, truncated K227Q Ligates the pre-adenylated 5´ end of DNA or RNA to the 3´ end of RNA. Mutation of K227 in T4 RNA Ligase 2 reduces enzyme lysyl adenylation. This mutation further reduces the formation of undesired ligation products (concatemers and circles) by T4 Rnl2tr, possibly by reducing the trace activity of T4 Rnl2tr in transfer of adenylyl groups from linkers to the 5´-phosphates of input RNAs. The exclusion of ATP, use of pre-adenylated linkers, and the reduced enzyme lysyl adenylation activity provide the lowest possible background in ligation reactions. This enzyme has been used for optimized linker ligation for the cloning of microRNAs. https://www.neb.com/en/products/m0351-t4-rna-ligase-2-truncated-k227q#Product%20Information 07/10/2024 18  T7 RNA polymerase T7 RNA Polymerase T7 RNA Polymerase is used for in vitro mRNA synthesis and is highly specific for the T7 phage promoter. Applications include: Radiolabeled RNA probe preparation RNA generation for studies of RNA structure, processing and catalysis Expression control via anti-sense RNA mRNA, sgRNA synthesis https://www.neb.com/en/products/m0251-t7-rna-polymerase#Product%20Information 07/10/2024 19  CircLigase A thermostable TS2126 RNA ligase derived from the Thermus bacteriophage TS2126 that infects the thermophilic bacterium, Thermus scotoductus Has been sold as THERMOPHAGE RNA ligase II or THERMOPHAGE ssDNA ligase by Prokaria Now known as Circligase by Epicenter, supplied by Lucigen. Template-independent circularization of linear ssDNA to circular ssDNA TS2126 RNA ligase is significantly more thermostable than those of mesophilic RNA ligases such as the T4 RNA ligase stable at up to 75 degrees C reduce undesirable secondary structures of ssDNA. CircLigase I thermostable ATP-dependent ligase CircLigase II ATP-independent Patent EP2396430A1, US9217167B2 https://shop.biosearchtech.com/lucigen 07/10/2024 20 Break 07/10/2024 21 Biotechnology 07/10/2024 22  tRNAs in biotechnology Tandemly arrayed tRNA-gRNA has enabled simultaneous tRNA sequencing has been used to profile the Production of non-natural proteins. gRNA expression and editing in various species. gut microbiome in mice, similar to traditional 16S ribosomal RNA sequencing. Su, Z., Wilson, B., Kumar, P. & Dutta, A. Noncanonical Roles of tRNAs: TRNA Fragments and beyond. Annu. Rev. Genet. 54, 47–69 (2020). 07/10/2024 23 Viral tRNAs are expressed during infection Host tRNA Host tRNA Viral tRNA 07/10/2024 24 Nyerges, A. et al. A swapped genetic code prevents viral infections and gene transfer. Nature 615, 720–727 (2023).  tRNAs in biotechnology b, Schematic of viral infection in Ec_Syn61Δ3-SL. The reassignment of the sense codons TCA and TCG to leucine in Ec_Syn61Δ3-SL provides multi-virus resistance by mistranslating the viral proteome. 07/10/2024 25 Nyerges, A. et al. A swapped genetic code prevents viral infections and gene transfer. Nature 615, 720–727 (2023).  IRES - internal ribosome entry site Strong inducers of translation initiation Initiate translation in a cap-independent fashion by binding to the ribosome IRES sequences are present in many native organism genes Virus genes use IRES sequences effectively to hijack the translation machinery of the cell Viral IRES’s are considerably better at initiating translation compared to their cellular IRES counterparts as a result of evolutionary pressure IRESs are attractive for combined transcription and translation (Tx/Tl) CFPS reactions C. Eric Hodgman, Michael C. Jewett, Characterizing IGR IRES-mediated translation initiation for use in yeast cell-free protein synthesis, New Biotechnology, Volume 31, Issue 5, 2014,Pages 499-505, 07/10/2024 26 ISSN 1871-6784, https://doi.org/10.1016/j.nbt.2014.07.001.  mRNA in biotech RNA vaccines Deliver the transcript of interest, encoding one or more immunogen(s), into the host cell cytoplasm. Manufacturing process includes Plasmid DNA (pDNA) DNA-dependent RNA polymerase promoter, such as T7 5′ cap and the 3′ poly(A) tail Purification with high-pressure liquid chromatography (HPLC) 07/10/2024 27 mRNA in biotech RNA vaccines 07/10/2024 28  mRNA in biotech RNA vaccines Two categories of mRNA constructs are being actively evaluated: 1. Non-replicating mRNA (NRM) 2. Self-amplifying mRNA (SAM) constructs 07/10/2024 29 Mechanism of action for NRM and SAM. Jackson, N.a.C., Kester, K.E., Casimiro, D., Gurunathan, S., and Derosa, F. (2020). The promise of mRNA vaccines: a biotech and industrial perspective. npj Vaccines 5. 07/10/2024 30 Key components of mRNA for expressing gene of interest. Jackson, N.a.C., Kester, K.E., Casimiro, D., Gurunathan, S., and Derosa, F. (2020). The promise of mRNA vaccines: a biotech and industrial perspective. npj Vaccines 5. 07/10/2024 31 mRNA vaccines in development. 07/10/2024 32 Small RNAs in biotech https://www.biotech-careers.org/articles/why-non-coding-rna-hot-biotechnology 07/10/2024 33 Small RNAs in therapeutics Monica R. Lares et, al. 07/10/2024 34 miRNA in diagnostics Phuong T. B. Ho er, al. 07/10/2024 35  How to avoid RNase contamination https://www.neb.com/en/tools-and-resources/video-library/avoiding-rnase-contamination?autoplay=1 07/10/2024 36  What to expect from the exam Figures Questions 07/10/2024 37  Seminar 6 Homework Everyone reads the paper: On-demand biomanufacturing of protective conjugate vaccines Stark, J. C. et al. On-demand biomanufacturing of protective conjugate vaccines. Sci. Adv. 7, eabe9444 (2021). Group 6 Presents the background, introduces the paper and shows the gap in the field. Explains the common terms and methods used in the paper. Presents the first figure and starts the discussion. Group 6 will facilitate discussion and moderate of discussion. Group 6 will explain the 7th figure (summary figure) of the paper Other groups Will explain the rest of the figures by group in a random order. We will all go over what was learned during the course Prepare questions if something was unclear 07/10/2024 38