In vitro RNA

Fernandez A and Cendra H 1996 in vitro RNA folding the principle of sequential minimization of entropy loss at work Biophys. Chem. 58 335-9... 

The apparent molecular weights of both natural P. pyralis luciferase and an active luciferase obtained from P. pyralis by the in vitro RNA translation were 62,000 by SDS-PAGE (Wood et al., 1984), in contrast to the value of 100,000 that had been widely referred to in the field for almost 30 years. Luciferases from other species of firefly probably have similar molecular weights. Presently, the molecular masses of firefly luciferases are considered to be 60-62 kDa. 

Subsequent to DNA template linearization, the procedures for in vitro RNA transcription reaction are set up. It is essential to avoid RNase contamination by using gloves, sterile glassware, and water devoid of RNase activity [treated with 0.1% diethyl pyrocarbonate (DEPC) and autoclaved]. 

Methot, N., Pickett, G., Keene, J.D. and Sonenberg, N. (1996) In vitro RNA selection identifies RNA ligands that specifically bind to eukaryiotic translation initiation factor 4B the role of the RNA recognition motif. RNA, 2, 38-50. 

Analyze the digestion by loading 10 fd of 7 fig/ml undigested and digested samples on a 1% agarose gel. The linearized plasmid now can be used directly to perform in vitro RNA transcription. 

Kern, J. A., and Davis, R. H. (1997). Application of solution equilibrium analysis to in vitro RNA transcription. Biotechnol. Prog. 13, 747-756. 

Johnston, D.E., Me Clure, W.R. Abortive initiation of in vitro RNA synthesis on bacteriophage X DNA. In RNA Polymerase. Cold Spring Harbor Laboratory 1976, p. 413... 

Dietary amaranth in animals results in an exfoliating or solubilizing effect on the brush border membrane of the small intestine. Amaranth stimulates in vitro RNA synthesis by causing the dissociation of chromatin. Amaranth has also been shown to increase kidney malate dehydrogenase activity after intramuscular dosing. 

RNA Polymerase slows down, or pauses, when it reaches the first GC-rich segment, because the stability of G-C base pairs makes the template hard to unwind. In vitro, RNA polymerase does pause for several minutes at a GC-rich segment. 

Montalvetti A, et al. (2003). Farnesyl pyrophosphate synthase is an essential enzyme in Trypanosoma brucei. In vitro RNA interference and in vivo inhibition studies. /. Biol. Chem. 278(19) 17075-17083. 

In addition to the five bases named adenine, guanine, cytosine, uracil, and thymine (3-methyl uracil), the polynucleotides also contain small amounts of their derivatives. Up to 6% of 5-methyl cytosine occurs in vegetable DNA and up to 1.5% of this occurs in certain mammalian DNA. In addition, 1-methyl guanine or dihydrouracil is found in RNA. These derivatives are produced in RNA by modification of the bases after the in vitro RNA synthesis. 

Such complexes are visible with the electron microscope and also may be detected by sucrose density gradient ultracentrifugation (Bytne et al., 1964). It has also been shown that the addition of ribosomes in the in vitro RNA polymerase system enhances RNA synthesis and the liberation of RNA from its complex with the template and the enzyme (Shin and Moldave, 1966 Jones et al., 1968). Thus in the bacterial cell the processes of transcription and translation usually are coupled (see Martin, 1969). 

Hatey F, Moule Y (1977) Inhibition of in vitro RNA and Protein Synthesis by Patulin. Ann Nutr Aliment 31 867... 

In addition to PCR, there are many other technologies to amplify nucleic acids. For example, ligation-based amplification or ligase chain reaction uses sequence-directed oligonucleotide primers and thermostable DNA ligase to assay point mutations, deletions, or insertions in DNA. Strand-displacement amplification uses the inherent strand-displacement activity of DNA polymerases to conduct DNA amplification at a constant temperature. Transcription-based methods such as nucleic acid sequence-based amplification (NASBA) involve in vitro RNA transcription. NASBA and most other transcription-based... 

Cytokinins have been suggested to play a critical role in several developmental processes in higher plants [5]. It is well documented that increases in RNA, protein and chlorophyll levels can be observed upon treatment with CKs [2, 6, 15], and, therefore, research on possible mechanisms of CK action in relation to CK-binding sites is logical. Although evidence has accumulated that CK-binding proteins can be isolated, and in fact do exist [3, 4, 14], and macromolecular factors that can interact with CKs and with in vitro RNA synthesis systems have been found [ 17,18], the specificities of these interactions remain to be established. 

In all of these experiments cytokinins stimulated in vitro RNA polymerase associated protein kinase activity. Thus, this enzyme may be one of the molecular targets for cytokinins. However the direct action of cytokinin on RNA polymerase in vitro activates only protein kinase without an effect on RNA polymerase. For revealing cytokinin effects on RNA polymerase activity in vitro, an additional cytokinin dependent cytoplasmic protein transcriptional factor was necessary [8]. 

Comparative studies [89] have shown that [(r -arene)Ru(en)Clj complexes, where arene = Bip, DHA, TH A or Cym bind relatively rapidly to calf thymus DNA at 310 K, with 50% binding in 3 h for the p-cymene complex and 10-15 min for the others. The major stop sites in assays of in vitro RNA synthesis by RNA polymerases on DNA templates modified by these Ru(II) arenes are similar to those exhibited by cisplatin-DNA adducts, although in general reduced relative to cisplatin. The efficiency of the p-cymene complex is noticeably lower than that of the other complexes. 

Rifampicin is also an inhibitor of the synthesis of a number of phages and viruses. It has been demonstrated that the RNA polymerase, which transcribes phage p 22 following infection of Bacillus subtilis, retains the rifampicin sensitivity of the host cell enzyme , Rifampicin also inhibits the formation of infectious vaccinia virus and viral particles. Whereas virion formation is completely inhibited neither the synthesis of RNA and protein nor the activity of in vitro RNA polymerase associated to the virion is affected lt>, Rifampicin inhibits the multiplication of poxvirus in vitro and in vivo. The side chain of this antibiotic derivative appears to be essential for the anti-viral effect and anti-trachomal activity found in... 

The discovery of the ribozyme sparked new debate on the RNA world hypothesis, where all biological processes were carried out by RNA-based enzymes. Since then, RNA evolution has been forced in vitro to come up with RNA enzymes capable of carrying out a wide variety of biochemical reactions, as far-reaching as carbon—carbon bond and peptide bond formation. In vitro RNA evolution... 

When RNA is isolated from infected cells, however, such a polycistronic messenger is not found (Summers, I969 Siegel and Summers, 1970). Instead discrete species of smaller sizes are found. A posttranscriptional modification mechanism in vivo has been proposed which would allow the production of smaller species (Millette et al., 1970). Very recent experiments have produced evidence that such posttranscriptional cleavage indeed exists (Dunn and Studier, 1973 a). RNase III from uninfected cells cleaves the in vitro RNA into just those 5 main RNA species which occur in vivo after T7 gene 1... 

Fig. 12. Dependence on template of RNA directed enzyme synthesis in vitro, A DEAE system of the composition described in Table 2 was started with varying amounts of RNA per incubation mixture. The isolation of in vivo RNA (in this case 10 minutes past T7+ infection) has been described (Herrlich and Schweiger, 1974), for the preparation of in vitro RNA (by E. coli RNA polymerase on T7 DNA) see Schweiger...

Davis, R. W., Hyman, R. W. Physical locations of the in vitro RNA initiation site and termination sites of T7M DNA. Cold Spr. Harb. Symp. quant. Biol. 35,269-281 (1970). 

When effective, 90% or more of a gene s expression can be suppressed. However, determining an effective siRNA sequence can be a trial and error approach. Thus, the work can become time and resource intensive as the iterations accumulate. To address this, in vitro RNA synthesis can be done that exploits the T7 promoter and T7 RNA polymerase. A sense DNA strand that encodes a candidate sequence, which has eight bases that are complementary to the 3 end of T7 promoter, is first hybridized with the T7 promoter. A full duplex then is synthesized with the help of Klenow DNA polymerase. Transcription by T7 RNA polymerase then is performed to produce many RNA copies. The same procedure is done... 

---