Modification interference

Green L, Waugh S, Binkley JP, Hostomska Z, Hostomsky Z, Tuerk C. Comprehensive chemical modification interference and nucleotide substitution analysis of an RNA pseudoknot inhibitor to HIV-1 reverse transcriptase. J Mol Biol 1995 247 60-68. 

The basis of a modification interference experiment is an initial modification followed by a selection procedure, where RNA molecules that retain their normal properties with respect to the selection procedure are separated from molecules with altered properties (e.g. modified RNA molecules that still bind a protein can be separated from modified RNA molecules that are unable to bind the protein see Fig. 4.15). 

Hydrazine, CMCT, kethoxal, DMS and DEP (described in Section 4.3.2) are useful for modification interference experiments. Procedures in Section 4.4.2 can be adopted directly for modifications under native conditions, while the procedures used for chemical sequencing of RNA (Section 4.4.3.2.2) generally produce a uniformly modified RNA at denaturing conditions. 

Similar assays can be used to study modifications that inhibit the extension of the reverse transcriptase directly and also modifications which render the RNA susceptible to selective strand scission at the modified residue (e.g. m7 guanosines, Section 4.4.2.2). To quantify the extent of modification, one of the nucleotides in the extension mixture should only be present in the dideoxy form (Fig. 5.7). The primer extension assay can also be used for some modifications which do not affect the extension, provided the modification interferes with the reactivity of the nucleotide. The latter approach has been used to study the 2 -0-methylations of guanosine where the modification renders the modified guanosine resistant to RNase T1 digestion. Introduction of a complete RNase T1 digest before the primer extension allows the detection of the modification (Fig. 5.8a). However, the approach requires that there is no nucleotide as the one modified for at least 12 nucleotides 3 of the modified yresidue. If this is not the case alternative approaches can sometimes be employed (see Fig. 5.8b). 

The relative binding affinities for the VDR were slightly reduced as compared to the natural hormone calcitriol. Thus, the side-chain modifications interfere only marginally with VDR binding (Table 10.5) [8],... 

Fig. 18.20. Left In vitro binding site of the basic subdomain of human immunodeficiency virus type-1 (HIV-1) transactivator protein (Tat) in the transactivation response (TAR) RNA sequence. Only a short portion of the stem region of TAR is shown. Sites where residue modification interferes with Tat binding (the putative binding site) are bold. They comprise part of the pyrimidine bulge and the adjacent duplex, but not the hairpin loop, shown at the top. The boxed base U24 enhances binding when eliminated, whereas modification of C23 does not interfere with peptide binding. The dashed line connecting C and G in the six-base loop indicates the possible formation of a base pair.

An homologous cell free system which utilizes synthetic SL RNA (generated by in vitro transcription) in trans-splicing has permitted a detailed dissection of the nematode SL RNA (34). Site-directed mutagenesis and chemical modification interference studies have revealed that critical functional elements in the SL RNA are confined to remarkably short regions of the molecule, all of which reside in the snRNA-like domain... 

The subtle interaction of air pollutants with these other stressors to plants and vegetation is the subject of ongoing research. For some plant systems, exposure to air pollutants may induce biochemical modifications which interfere with the water balance in plants, thereby reducing their ability to tolerate drought conditions. 

Pre-column off-line derivatisation requires no modification to the instrument and, compared with the post-column techniques, imposes fewer limitations on the reaction conditions. Disadvantages are that the presence of excess reagent and by-products may interfere with the separation, whilst the group introduced into the molecules may change the chromatographic properties of the sample. 

NOTE The test method is accurate down to 2 ppb, but some colorimetric interference may be noted, thus requiring a modification to the method. 

Furthermore, there is the problem that the signal level to whieh one extrapolates need not necessarily be y = 0 if there is any interference by a matrix component, one would have to extrapolate to a level y > 0. This uncertainty can only be cleared if the standard addition line perfectly coincides with the calibration line obtained for the pure analyte in absence of the matrix, i.e. same slope and 100% recovery, see also Figure 3.2. This problem is extensively treated in Refs. 97-101. A modification is presented in Ref. 102. 

Electrical outlets can be installed in one of several ways. Small pedestals holding two or more outlets are often placed along the rear of wall-mounted work benches. They may also be put along the center of peninsulas or islands. Wiring is from below the countertop, which makes modifications difficult, just as with plumbing. While easy to reach, pedestals clutter the top and interfere with cleaning. 

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