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The Emerging Field of RNA Nanotechnology
Published in Lajos P. Balogh, Nano-Enabled Medical Applications, 2020
For fluorescent labelling, single conjugation of fluorophores at the 5′ - or 3′ -end is preferable to prevent physical hindrance. End-labelling is not difficult with chemical synthesis of small RNA, however, it is challenging for long RNA requiring enzymatic methods. To meet this challenge, GMP or AMP derivatives that can only be used for transcription initiation, but not for chain elongation, have been used. Fluorescent RNA can also be easily synthesized in vitro with T7 RNA polymerase using a new agent tCTP [114].
Evolution
Published in Paul Pumpens, Single-Stranded RNA Phages, 2020
Rather suddenly, Biebricher and Luce (1996) discovered the template-free generation of RNA species that were replicated with the phage T7 RNA polymerase by incubating high concentrations of this enzyme with substrate for extended time periods. The products differed from sample to sample in sequence and length ranging from 60−120 nucleotides. The authors regarded the mechanism of autocatalytic amplification of RNA by T7 RNA polymerase to be analogous to that observed with the Qβ replicases. With enzyme in excess, exponential growth was observed; linear growth resulted when the enzyme was saturated by RNA template. The secondary structures of all species sequenced turned out to be hairpins. The RNA species were not accepted as templates by the Qβ replicase, or by the RNA polymerases from E. coli or phage SP6, while T3 RNA polymerase was partially active. The template-free RNA production was completely suppressed by addition of DNA to the incubation mixture. No replicating RNA species were detected in vivo in cells expressing the T7 RNA polymerase (Biebricher and Luce 1996). This finding indicated a wider occurrence of the RNA replication than previously assumed. Biebricher and Luce (1996) pointed to viroids, the smallest agents of infectious diseases known, as the remarkable natural example of a parasitic RNA directing its own replication by a host RNA polymerase (Diener et al. 1993).
Gene Expression
Published in Danilo D. Lasic, LIPOSOMES in GENE DELIVERY, 2019
Some results with nonviral lipid delivery systems show that only a small fraction (~1%) of DNA introduced into the cell cytoplasm actually reaches the nucleus. There, DNA must be transcribed into mRNA which, upon further changes, migrates into cytoplasm to ribosomes where protein synthesis commences (Chen et al., 1993). It may be advantageous if gene expression would begin in the cytoplasm. Indeed, some vectors, such as bacteriophage T7 which encodes for an enzyme T7 RNA polymerase which can transcribe DNA into RNA in the cytosol of mammalian cells with high transcriptional activity, can express cDNA in the cytoplasm. For such a system to work, a T7 RNA polymerase protein must be present in the cytoplasm along with the cDNA of interest. It can be introduced together with DNA, encoded in the DNA, or both. Expression of both genes is induced by T7 RNA polymerase promoters and rapid cytoplasmic gene expression independent of nuclear transcription factors was observed. Transcripts are not capped and an IRES sequence is inserted into the 5’UTR for efficient translation of transcripts. Although high levels of expression can be achieved, it is only transient because polymerase is degraded. By using a T7 autogene, however, a continuous synthesis of T7 RNA polymerase can be achieved, and expression for a week was reported (Gao and Huang, 1993). Such an approach may be useful when a fast, transient, and high level of transgene expression is preferred.
Vaccines on demand, part II: future reality
Published in Expert Opinion on Drug Discovery, 2023
Andrew J. Geall, Zoltan Kis, Jeffrey B. Ulmer
The RNA component of both mRNA and saRNA vaccines is synthesized using a cell-free in vitro transcription reaction, which can be completed in 2 hours [1,40–42]. Thereby, the T7 RNA polymerase creates the RNA based on a template DNA. Following synthesis, the RNA is purified using one or more chromatography unit operations and usually several tangential flow ultrafiltration and diafiltration unit operations. Removal of product-related impurities (e.g. double-stranded RNA and partially degraded RNA) can be challenging, however the following chromatography techniques can be used for this: oligo(dT) affinity, reverse-phase, hydrophobic interaction, ion exchange, and multi-modal chromatography [1,21,40–42]. After downstream purification, the RNA is formulated into lipid nanoparticles (LNPs) using microfluidics equipment [43,44], impingement jet mixers [45], T-junction mixers [44], multi-inlet vortex mixers [46] or pressurized tanks [47]. The LNP-encapsulated RNA is then purified for example, by tangential flow ultrafiltration and diafiltration. Next, the RNA-LNP solution is sent for fill-finish. However, the fill-finish processes as well as the plasmid DNA production, purification, and linearization were not modeled in this study.
CRISPR-based biosensing systems: a way to rapidly diagnose COVID-19
Published in Critical Reviews in Clinical Laboratory Sciences, 2021
Majid Vatankhah, Amir Azizi, Anahita Sanajouyan Langeroudi, Sajad Ataei Azimi, Imaneh Khorsand, Mohammad Amin Kerachian, Jamshid Motaei
The CREST(Cas13-based, rugged, equitable, scalable testing) platform may be able to perform large-scale COVID-19 testing utilizing a miniPCR thermocycler and low-cost reagents without losing sensitivity. The CREST platform was created using the nonspecific collateral cleavage activity of the Cas13 effector. After reverse transcription, the RNA was amplified by PCR using Taq polymerase enzyme, which is widely available. Then, the DNA was transcribed by T7 RNA polymerase to provide RNA substrates for Cas13a. Following the identification of the target sequence in the RNA, Cas13-gRNA complex cut the reporter molecules and released the signal. In this platform, a P51 molecular fluorescence visualizer was used instead of an immunochromatography assay to detect the results. CREST has been shown to be as sensitive as RT-qPCR, to have less costly reagents, and to have upfront instrument costs that are 30–50 times less expensive. The LoD of the CREST protocol was 10 copies/µL input of the RNA SARS-CoV-2. CREST was also more sensitive and cost-effective than the Cas13 diagnostic platforms using amplification with RT-RPA or RT-PCR [71].
Ravaging SARS-CoV-2: rudimentary diagnosis and puzzling immunological responses
Published in Current Medical Research and Opinion, 2021
Tapan Kumar Mukherjee, Parth Malik, Radhashree Maitra, John R. Hoidal
Developed in 2017, the SHERLOCK technique aims at Cas13a facilitated highly sensitive rapid nucleic acid detection with single base specificity, for exclusive binding and cleavage of COVID-19 single stranded RNA48–51. Figure 3(a) depicts the SHERLOCK procedure, wherein RNA is isolated from viral population residing in the nasopharyngeal swabs of the patients followed by c-DNA preparation from RPA. The in vitro transcription is performed with T7 RNA polymerase after which the newly prepared samples are exposed to the Cas13 detection system. Viral transcripts if present activate the cleavage reporter molecule, thereby aiding the cleaved reporter detection using dipstick technique within one hour. The term SHERLOCK signifies a moveable platform, which in combination with isothermal Cas13, assisted pre-amplification besides RNA or DNA screening52.