Analogue interference

An appropriate selection procedure is applied to the RNA transcript containing the modified residues. 

The selected pool of RNA is then cleaved quantitatively at the modified residues by iodine as described in Section 4.4.2.7 and analysed by polyacrylamid gel electrophoresis. 

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Oyelere AK, Kardon JR, Strobel SA. pK(a) perturbation in genomic Hepatitis Delta Virus ribozyme catalysis evidenced by nucleotide analogue interference mapping. Biochemistry 2002 41 3667-3675. 

Random incorporation of modified residues in in vitro transcribed RNA is of particular interest in analogue interference experiments (Section 4.6.3) where randomly modified RNA molecules with altered properties are selected and compared to the original pool of RNA molecules. A crucial prerequisite for the interference analysis is the ability to identify the modified residues. A convenient and efficient method is the use of nucleotides which contain both the modification of interest (e.g. 2 -0-methyl NTP, dNTP, inosinetri-phosphates) and an a-phosphorothioate group (one of the nonbridging oxygens is replaced with a sulphur).14-16 The concurrent incorporation of the phosphorothioate renders the modified nucleotides sensitive to a selective cleavage by iodine. 

No analogue has yet been found which interrupts chain elongation by causing a block in peptide-bond formation. Such a situation seems to be highly improbable. Examples of premature arrest in chain elongation are known, but are the result of secondary effects arising from analogue interference. 

NAIM/NAIS nucleotide analogue interference mapping/suppression... 

Dihydro compounds show reactions which parallel those of their aliphatic analogues provided that the aromatization reactions just discussed do not interfere. 

Antimetabolites interfere with normal metabolic pathways. They can be grouped into folate antagonists and analogues of purine or pyrimidine bases. Their action is limited to the S-phase of the cell cycle and therefore they target a smaller fraction of cells as compared with alkylating agents. 

In 1985, it was reported by Hsiang et al. [43] that the cytotoxic activity of 20-(S)-camptothecin (CPT III) was attributed to a novel mechanism of action involving the nuclear enzyme topo I, and this discovery of unique mechanism of action revived the interest in CPT and its analogues as anticancer agents. CPT stabilizes the covalent, reversible topo I-DNA complex leading to the inhibition of DNA synthesis in mammalian cells and interferes with the topo I breakage-reunion reaction [44]. Clinical trials and structure-activity relationships have demonstrated the requirement of the a-hydroxy group, the... 

Salicylic acid analogues are often active as nonsteroidal antiinflammatory agents because they interfere with biosynthesis of prostaglandins. Diflunisal (3) appears to be such an agent. It is synthesized from the nitrobiphenyl 1 by catalytic reduction to... 

An antimetabolite interferes with the normal cellular metabolites. For instance, it can act as an inhibitor of one or more enzymes whose substrates are metabolites. Others are incorporated into macromolecules instead of the metabolites. Development of antimetabolites exhibiting anti-cancer activity met with the greatest success for analogues of metabolites involved in the biosynthesis of nucleic acids and of cofactors containing nitrogenous bases. Compounds such as 5-fluorouracyl and methotrexate are remarkably effective against human cancers, even though they feature host toxicity. 

Comment It is critical to remove excess cap analogue from in vitro transcribed transcripts prior to transfection, because the cap analogue will compete with transcripts for the cellular translational machinery. Also, even after DNase treatment, the RNA sample may still contain traces of functional pDNA, which may interfere with subsequent detection by RT-PCR. Furthermore, plasmids containing a mammalian promoter may even give rise to de novo transcription in transfected cells. 

The analogue between the Young- interferometer (interference pattern in the spatial domain) and the serrodyne modulated MZI (interference pattern in the time domain) is striking. An optimized Young interferometer17 can also show resolutions down to <3n 10 8. 

In order to evaluate the individual properties as qubit of each Ln(III) ion within the pertinent [Ln2] complexes, it is necessary to study each ion without the interference of the other. This could be done if it were possible to prepare dinuclear complexes containing the l.n(III) to be studied in the desired position accompanied by a diamagnetic Ln(III) centre (or analogue) at the other site. This was possible thanks to the exceptional structural characteristics of the [Ln2] family. 

Even nowadays the application of radioactive isotopes is the most sensitive method for the analysis of biomolecules or their reaction products. Besides the low detection limits, the replacement of a naturally overbalancing stable isotope by its radioactive analogue does not interfere with the physical or chemical properties of the enzyme (with some exceptions for hydrogens). Figure 6 lists some frequently used radioactive isotopes and their half-life periods. 

In the U.S.A. various pyridazine analogues of naturally occurring pyrimidine nucleosides have been prepared [299, 300] for a review on this subject see [13]. Within this series, 3-deaza-6-azauridine (86) has been found to inhibit the growth of L-1210 mouse leukaemic cells with an ID50 value of a 7 X 10 5 M [299, 301], The inhibitory effect of this uridine isoster might be due to interference in pyrimidine biosynthesis [302],... 

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