Ribonuclease activity of cationic structures conjugated to lipophilic groups

Cationic compounds containing benzene ring substituted with the bis-quaternary salt of diazabicyclo[2.2.2]octane (DABCO) bearing a polymethylene fragment at the bridge positions display ribonuclease activity. Efficacy of the catalysis is affected by geometry of the cationic structures and the size of the attached aliphatic fragment. The cleavage occurs primarily within CA sequences. The compounds do not possess tradition groups participating in the transesterification step of RNA cleavage reaction, therefore a speculative mechanism of cleavage could be inducing a conformational stress on the RNA sugar phosphate backbone providing fragility to phosphodiester bonds.     

Specific cleavage of ribosomal RNA caused by alpha sarcin.

Alpha sarcin causes the specific cleavage of RNA from 80S ribosomes and 60S subunits of yeast, but not from the 40S subunits to produce a small RNA fragment. The fragment was also produced on treatment of the 60S subunits of wheat germ ribosomes. The fragment has a molecular weight of 100,000 and is a cleavage product of the large RNA species in the 60S subunits. The fragment is not derived from the 5'end of the yeast 25S RNA nor does it bind to 5.8S RNA and we propose that the fragment represents the 3' terminal 320 nucleotides of 25S rRNA. The ability to produce fragment could not be separated from the ability of alpha sarcin to inhibit protein synthesis. Alpha sarcin also causes the specific cleavage of the 23S RNA of the E. coli subunit to produce a smaller fragment of RNA than that produced from eukaryote ribosomes.     

Self-cleavage of p2Sp1 RNA with Mg2+ and non-ionic detergent (Brij 58)

The precursor of an RNA molecule from T4-infected E. coli cells (p2Sp1 RNA) has the capacity to cleave itself at specific positions [(UpA (139-140) and CpA (170-171)], within a putative loop and stem structure. This sequence-specific cleavage requires at least a monovalent cation and non-ionic detergents. We studied the self-cleavage reaction of an RNA fragment (GUUUCGUACAAAC) (R1) with the sequence corresponding to the p2Sp1 RNA in the presence of Mg2+ and non-ionic detergents. It requires Mg2+ and is aided by a non-ionic detergent, Brij 58. The cleavage reaction is time, temperature, and pH-dependent. The cleavage occurs at the phosphodiester bond between UpA and CpA on the RNA fragment (GUUUCGUACAAAC) (R1). Furthermore, the maximum of cleavage of R1 occurs at a very low Mg2+ concentration (< or = 5 mM).     

Characterization of deoxy- and ribo-containing oligonucleotide substrates in the hammerhead self-cleavage reaction

Deoxynucleotides were introduced into a substrate fragment of the hammerhead RNA self-cleaving domain. A substrate lacking the 2' hydroxyl adjacent to the cleavage site showed no detectable cleavage under a variety of reaction conditions. Competition experiments indicate that this fragment binds to the ribozyme with an affinity similar to the all RNA fragment, suggesting that the attacking 2' hydroxyl does not substantially contribute to the binding of substrate to ribozyme. Similar competition experiments with the all DNA substrate indicate a much lower affinity for the ribozyme perhaps due to the lack of other 2' hydroxyls. A substrate containing all deoxy residues except for a ribonucleotide at the cleavage site was also shown to be active.     

The use of hydroxylamine cleavage to produce a fragment of ribosomal protein S4 which retains the capacity to specifically bind 16S ribosomal RNA.

In previous reports we have described the isolation of fragments of 30S ribosomal protein S4 using a number of different enzymatic and chemical cleavage techniques. These experiments were designed to determine the region of the protein responsible for 16S RNA recognition. We report here the isolation of two fragments produced by the hydroxylamine cleavage of the asparaginyl-glycyl peptide bond between positions 124 and 125. The purified fragments were chemically identified and tested for RNA binding capacity. The fragment consisting of residues 1-124 retains RNA binding activity and the fragment 125-203 is totally without RNA binding function. These results and previous results strongly suggest that the domain of protein S4 responsible for 16S RNA specific association is within the region consisting of residues 46-124.     

Site-Specific Cleavage of the Host Poly(A) Binding Protein by the Encephalomyocarditis Virus 3C Proteinase Stimulates Viral Replication

Although picornavirus RNA genomes contain a 3'-terminal poly(A) tract that is critical for their replication, the impact of encephalomyocarditis virus (EMCV) infection on the host poly(A)-binding protein (PABP) remains unknown. Here, we establish that EMCV infection stimulates site-specific PABP proteolysis, resulting in accumulation of a 45-kDa N-terminal PABP fragment in virus-infected cells. Expression of a functional EMCV 3C proteinase was necessary and sufficient to stimulate PABP cleavage in uninfected cells, and bacterially expressed 3C cleaved recombinant PABP in vitro in the absence of any virus-encoded or eukaryotic cellular cofactors. N-terminal sequencing of the resulting C-terminal PABP fragment identified a 3C(pro) cleavage site on PABP between amino acids Q437 and G438, severing the C-terminal protein-interacting domain from the N-terminal RNA binding fragment. Single amino acid substitution mutants with changes at Q437 were resistant to 3C(pro) cleavage in vitro and in vivo, validating that this is the sole detectable PABP cleavage site. Finally, while ongoing protein synthesis was not detectably altered in EMCV-infected cells expressing a cleavage-resistant PABP variant, viral RNA synthesis and infectious virus production were both reduced. Together, these results establish that the EMCV 3C proteinase mediates site-specific PABP cleavage and demonstrate that PABP cleavage by 3C regulates EMCV replication.     

Cleavage of RNA by an amphiphilic compound lacking traditional catalytic groups

Recently, in experiments with combinatorial libraries of amphiphilic compounds lacking groups, known as catalysts of transesterification reaction, we discovered novel RNA-cleaving compounds [N. Kovalev, E. Burakova, V. Silnikov, M. Zenkova, V. Vlassov, Bioorg. Chem. 34 (2006) 274-286]. In the present study, we investigate cleavage of RNA by the most active representative of these libraries, compound named Dp12. Sequence-specificity of RNA cleavage and influence of reaction conditions on cleavage rate suggested that Dp12 enormously accelerates spontaneous RNA cleavage. Light scattering experiments revealed that the RNA cleavage proceeds within multiplexes formed by assembles of RNA and Dp12 molecules, at Dp12 concentration far below critical concentration of micelle formation. Under these conditions, Dp12 is presented in the solution as individual molecules, but addition of RNA to this solution triggers formation of the multiplexes. The obtained data suggest a possible mechanism of RNA cleavage, which includes interaction of the compound with RNA sugar-phosphate backbone resulting in changing of ribose conformation. This leads to juxtaposition of the 2′-hydroxyl group and internucleotide phosphorus atom at a distance needed for the transesterification to occur.     

Cleavage properties of an oligonucleotide containing a bridged internucleotide 5'-phosphorothioate RNA linkage.

An oligonucleotide has been synthesized that contains a single bridging 5'-phosphorothioate at an RNA linkage (5'-ApCpGpGpTpCpTprCpsApCpGpApGpC-3'). This new phosphodiester linkage is found to be particularly susceptible to cleavage when compared with the corresponding oxo, deoxy and thiodeoxy derivatives. Divalent metal cations were observed to dramatically increase the cleavage rate. The products of the cleavage under a variety of conditions are a 5'-thiol-containing fragment (6mer) and a 2',3'-cyclic phosphate-containing fragment (8mer). The pseudo-first order rate constant, kobs, for cleavage at pH 7.5 (50 mM Tris-HCI) in the presence of 5 mM EDTA is 1.5 x 10(-4)/min. In the presence of 5 mM metal dichloride and 50 mM Tris-HCI, pH 7.5, the relative cleavage rate enhancements are 10, 24, 71, 98, 370 and 3400 for Mg2+, Ca2+, Mn2+, Co2+, Zn2+ and Cd2+ respectively. The rate enhancements correlate well with Pearson's HSAB principle, suggesting that cleavage is mediated in part by coordination of the metal to the 5'-mercapto leaving group. RNA linkages containing bridging 5'-phosphorothioates should prove valuable for studying the mechanistic details of a variety of RNA cleaving agents, such as ribozymes.     

The stem hairpin loop structure of p2Sp1 RNA is required for RNA-cleaving activity

We studied the hairpin-loop structure of an RNA fragment (GUUUCGUACAAAC) (R13) with the sequence corresponding to the self-cleavage domain in the precursor of an RNA molecule from bacteriophage T4-infected Escherichia coli cells (p2Sp1 RNA). In order to determine the influence of the hairpin-loop structure on these sequence-specific cleavage reactions, we have synthesized oligoribonucleotides containing hairpin-loop, double-helical stem-loop, and single-stranded RNA structures. The cleavage was affected by the hairpin-loop structure. Furthermore, the helix-stem, which retains the thermodynamically extrastable stem hairpin-loop structures, is also important for the cleavage activity. However, the thermodynamically extrastable helix-stem structure reduced the cleavage activity of the adjacent UA and CA sequences at the helix-stem site. For the cleavage reactions of the RNA cleavage products, the R6 (ACAAAC), R7 (GUUUCGU), and R9 (GUUUCGUAC) mers from the parent RNA, R13 (GUUUCGUACAAAC), a very slight amount of cleavage product (2%) from the RNA 9 was observed, but no reaction occurred for the R6 and R7. We also describe the influences of the sequences (UA and CA) on the cleavage activity.     

RNase activity of a DNA minor groove binder with a minimalist catalytic motif from RNase A

Imidazole and compounds containing imidazole residues have been shown to cleave RNA in an RNase A-mimicking manner. Di-imidazole lexitropsin is a compound which is derived from the polyamide drugs distamycin and netropsin essentially by the replacement of two pyrrole heterocycles with N-methyl-imidazole residues. This enables it to bind to the minor groove of B-DNA in a sequence-specific manner. We demonstrate here that this lexitropsin derivative has RNA cleavage activity, as tested on model RNAs. Optimal cleavage conditions and cleavage specificity resemble those known from other imidazole conjugates and are thus consistent with an RNase A type cleavage mechanism. The optimum concentration of the compound for cleavage is similar to previously investigated imidazole-based RNase mimics. As a whole new class of chemical compounds capable of interacting with nucleic acids through extensive hydrogen bonding, these imidazole containing compounds constitute promising scaffolds and ligands, for the construction of novel RNase mimics with high affinity.     

The kinetics of colicin E3 induced fragmentation of Escherichia coli 16S ribosomal RNA in vivo

Fixation of colicin E3 to sensitive bacteria is followed, after a lag of 2 to 6 min, by the rapid degradation of all the RNA of the 30S ribosomal subunits, yielding a large 15.5S fragment and a smaller fragment, containing the 3′-terminal end of the 16S RNA. The small RNA fragment which was estimated to consist of about 52 nucleotides, was retained within the 30S subunit in vivo and was subsequently recovered quantitatively without apparent further degradation. Kinetic studies of the cleavage of 16S RNA indicated that this is the primary and lethal effect of colicin E3 and the primary cause of the observed inhibition of protein synthesis in vivo. Small amounts of an RNA fragment, apparently identical in size to the small E3-fragment, were also isolated from 30S particles obtained from untreated bacteria.     

Chemical ribonucleases: VII. Effect of positively charged RNA-binding domains and hydrophobic fragments of the conjugates based on 1,4-diazabicyclo[2.2.2]octane and imidazol on their ribonuclease activity

The effect of the total positive charge in the RNA-binding domain of chemical ribonucleases that are conjugates of bisquaternary salts of diazabicyclo[2.2.2]octane and imidazol on the cleavage of an HIV-1 RNA fragment was studied. An increase in the positive charge from +2 to +4 was shown to result in a significant growth in the ribonuclease activity. Possible mechanisms of the interactions between structural moieties of chemical ribonucleases and RNA that enable an effective catalysis of the cleavage of phosphodiester bonds are discussed.     

Chemical ribonucleases: VII. Effect of positively charged RNA-binding domains and hydrophobic fragments of the conjugates based on 1,4-diazabicyclo[2.2.2]octane and imidazole on their ribonuclease activity

The effect of the total positive charge in the RNA-binding domain of chemical ribonucleases that are conjugates of bisquaternary salts of 1,4-diazabicylo[2.2.2]octane and imidazole on the cleavage of an HIV-1 RNA fragment was studied. An increase in the positive charge from +2 to +4 was shown to result in a significant growth in the ribonuclease activity. Possible mechanisms of the interactions between structural moieties of chemical ribonucleases and RNA that enable an effective catalysis of the cleavage of phosphodiester bonds are discussed.     

Pif1 helicase directs eukaryotic Okazaki fragments toward the two-nuclease cleavage pathway for primer removal

Eukaryotic Okazaki fragment maturation requires complete removal of the initiating RNA primer before ligation occurs. Polymerase delta (Pol delta) extends the upstream Okazaki fragment and displaces the 5'-end of the downstream primer into a single nucleotide flap, which is removed by FEN1 nuclease cleavage. This process is repeated until all RNA is removed. However, a small fraction of flaps escapes cleavage and grows long enough to be coated with RPA and requires the consecutive action of the Dna2 and FEN1 nucleases for processing. Here we tested whether RPA inhibits FEN1 cleavage of long flaps as proposed. Surprisingly, we determined that RPA binding to long flaps made dynamically by polymerase delta only slightly inhibited FEN1 cleavage, apparently obviating the need for Dna2. Therefore, we asked whether other relevant proteins promote long flap cleavage via the Dna2 pathway. The Pif1 helicase, implicated in Okazaki maturation from genetic studies, improved flap displacement and increased RPA inhibition of long flap cleavage by FEN1. These results suggest that Pif1 accelerates long flap growth, allowing RPA to bind before FEN1 can act, thereby inhibiting FEN1 cleavage. Therefore, Pif1 directs long flaps toward the two-nuclease pathway, requiring Dna2 cleavage for primer removal.     

DNA conjugates as novel functional oligonucleotides

Oligodeoxynucleotides with RNA cleavage activity 1) were conjugated with amines and peptides by solid phase fragment condensation (SPFC). It was found that 29 mer DNA enzyme conjugated with spermine at its 5'-end showed higher affinity to the target RNA sequence and 40 times higher activity of cleavage than native DNA enzyme. It is also to be noted that conjugate DNA enzymes showed increased resistance against nuclease digestion.