Site‐specific modification of 4.5S RNA apical domain by complementary oligodeoxynucleotides carrying an alkylating group

Abstract

Site-specific alkylation of RNA by reactive oligodeoxynucleotides provides structural information and represents the first step towards the design of RNA derivatives to be used for functional studies. Specific alkylation of 4.5S RNA at G53, the first base of the apical tetraloop, was achieved by incubation with oligodeoxynucleotide ON2, complementary to nucleotides 38Ϫ53, which carries a p- benzylamidophosphate group at the 5′ end. Alkylation efficiency was increased by a factor of 6, without alteration of specificity, in the presence of a helper oligodeoxynucleotide, ON1, complementary to nucleotides 58Ϫ71 of the opposite strand of the RNA helix. A second reactive oligodeoxynucleotide, ON1-3′-R, was obtained by attaching the alkylating group to the 3′ end of ON1. ON1-3′-R was able to modify G58. In the presence of ON2 as a helper oligodeoxynucleotide, the specificity of ON1-3′-R changes and efficient alkylation of nucleotides G54, A56 and G57 of the apical region of 4.5S RNA was observed.Keywords : Escherichia coli 4.5S RNA; RNA secondary structure ; site-specific modification ; reactive oligodeoxynucleotide.Interactions of RNA with other macromolecules are studied, complementary to the apical stem-loop structure of 4.5S RNA of Escherichia coli were shown to stimulate synergistically the among others, by cross-linking approaches. The most informative approaches are reactions of RNA molecules carrying indivi-formation of a ternary complex up to 10-fold (Malygin et al., 1996). Here, we use chemically reactive oligodeoxynucleotide dual activated bases. Derivatives of small RNAs can be synthesized chemically whereas methods to modify large RNA mole-derivatives bearing a p-(N-2-chloroethyl-N-methylamino)benzylamidophosphate group at either the 5′ or 3′ ends. After hybridcules at specific sites are still a challenging task. A very promising approach involves chemically reactive derivatives of anti-ization with 4.5S RNA, site-specific alkylation is observed.Upon combination with a second unmodified oligodeoxynucleosense oligonucleotides, among which phosphamide derivatives with a terminal alkylation group have proven to be most useful tide complementary to the opposite RNA strand, alkylation efficiencies are improved. In addition, a different hybrid structure (Zenkova et al., 1990(Zenkova et al., , 1995Venkstern et al., 1990). They are able to modify any nucleotide residue in RNA (except uridine) was observed in the presence of an oligodeoxynucleotide complementary to bases 38Ϫ53 of 4.5S RNA (ON2) as a helper neighboring the oligonucleotide 5′ or 3′ ends (Knorre et al., 1986). By this method, aminobenzyl groups are introduced, oligodeoxynucleotide which resulted in alkylation of another set of nucleotides within the apical domain of 4.5S RNA. which in turn can be converted into site-specific, photoreactive derivatives of RNAs. Modification efficiencies largely depend on the extent of hybridization between the target nucleic acid and the complementary oligodeoxynucleotide that often is inhib-MATERIALS AND METHODS ited...

Positioning of subdomain IIId and apical loop of domain II of the hepatitis C IRES on the human 40S ribosome

“…To increase the yield of IRES alkylation with the derivatives of deoxy-oligomers complementary to sequences 259–276 and 62–81, helper oligomers were used that facilitate binding of oligomers bearing the alkylating group to the structured RNA [see (34) and Refs. therein] (Figures 1 and 2b).…”

“…The oligos were 20-mers that was enough to enable them to form relatively stable heteroduplexes specifically with the target sequences, and contained an alkylating group at the 5′-termini. This group generally modifies RNA nucleotides near the 5′-end of the oligomer bound to the RNA, the more accessible is target RNA sequence, the higher is yield of the covalent adduct (34). To increase the yields of the covalent adducts, we used helper oligomers to disrupt the IRES secondary structure in the target region and thus to facilitate formation of the heteroduplexes with alkylating derivatives.…”

Proteins surrounding hairpin IIIe of the hepatitis C virus internal ribosome entry site on the human 40S ribosomal subunit

“…Alkylation of the IRES was carried out at 25°C for a period sufficiently long to almost completely convert the 2-chloroethylamine group to the ethylene immonium cation, which is an active intermediate ( 25 ). The ClRCH 2 NH- moiety coupled to the 5′-terminal nucleotide of the oligonucleotide is known to cross-link to the nucleotide base paired with it or to the neighboring unpaired nucleotide ( 17 , 26 ). Any RNA base at such positions may be cross-linked with the exception for uracil, which is unable to react with aromatic 2-chloroethyl amines under the conditions used ( 27 ).…”

Complementary-addressed site-directed spin labeling of long natural RNAs

“…In this approach, helper facilitates unfolding of RNA structure in the target sequence region and makes it accessible for binding with the oligonucleotide moiety of the DNA derivative (see Ref. ( 52 ) and Refs therein). With the use of helper complementary to sequence 84–102 (Figure 1 ), the extents of the alkylation of RNA by DNA1-NHCH 2 RCl and DNA2-NHCH 2 RCl were increased to approximately ∼0.5 and 0.6 mol of oligomer residue per mol of RNA, respectively (see also Supplementary Figure S1a).…”

“…The 4-[N-(2-chloroethyl)-N-methylamino]benzylphosphoramidе moiety attached to the 5′-terminal nucleotide of DNA might cross-link, as a rule, either to the last 3′-terminal RNA base in DNA•RNA heteroduplex or to the unpaired RNA base adjacent to it ( 52 – 54 ). In the former case, modification can occur only at atoms N7 of G or N3 of A that are not involved in the Watson–Crick base-pairing.…”