Position 34 of tRNA is a discriminative element for m5C38 modification by human DNMT2.

Dnmt2, a member of the DNA methyltransferase superfamily, catalyzes the formation of 5-methylcytosine at position 38 in the anticodon loop of tRNAs. Dnmt2 regulates many cellular biological processes, especially the production of tRNA-derived fragments and intergenerational transmission of paternal metabolic disorders to offspring. Moreover, Dnmt2 is closely related to human cancers. The tRNA substrates of mammalian Dnmt2s are mainly detected using bisulfite sequencing; however, we lack supporting biochemical data concerning their substrate specificity or recognition mechanism. Here, we deciphered the tRNA substrates of human DNMT2 (hDNMT2) as tRNAAsp(GUC), tRNAGly(GCC) and tRNAVal(AAC). Intriguingly, for tRNAAsp(GUC) and tRNAGly(GCC), G34 is the discriminator element; whereas for tRNAVal(AAC), the inosine modification at position 34 (I34), which is formed by the ADAT2/3 complex, is the prerequisite for hDNMT2 recognition. We showed that the C32U33(G/I)34N35 (C/U)36A37C38 motif in the anticodon loop, U11:A24 in the D stem, and the correct size of the variable loop are required for Dnmt2 recognition of substrate tRNAs. Furthermore, mammalian Dnmt2s possess a conserved tRNA recognition mechanism.

Anticodon-binding domain swapping in a nondiscriminating aspartyl-tRNA synthetase reveals contributions to tRNA specificity and catalytic activity.

The nondiscriminating aspartyl-tRNA synthetase (ND-AspRS), found in many archaea and bacteria, covalently attaches aspartic acid to tRNAAsp and tRNAAsn generating a correctly charged Asp-tRNAAsp and an erroneous Asp-tRNAAsn . This relaxed tRNA specificity is governed by interactions between the tRNA and the enzyme. In an effort to assess the contributions of the anticodon-binding domain to tRNA specificity, we constructed two chimeric enzymes, Chimera-D and Chimera-N, by replacing the native anticodon-binding domain in the Helicobacter pylori ND-AspRS with that of a discriminating AspRS (Chimera-D) and an asparaginyl-tRNA synthetase (AsnRS, Chimera-N), both from Escherichia coli. Both chimeric enzymes showed similar secondary structure compared to wild-type (WT) ND-AspRS and maintained the ability to form dimeric complexes in solution. Although less catalytically active than WT, Chimera-D was more discriminating as it aspartylated tRNAAsp over tRNAAsn with a specificity ratio of 7.0 compared to 2.9 for the WT enzyme. In contrast, Chimera-N exhibited low catalytic activity toward tRNAAsp and was unable to aspartylate tRNAAsn . The observed catalytic activities for the two chimeras correlate with their heterologous toxicity when expressed in E. coli. Molecular dynamics simulations show a reduced hydrogen bond network at the interface between the anticodon-binding domain and the catalytic domain in Chimera-N compared to Chimera-D or WT, explaining its lower stability and catalytic activity.

Bacterial Aspartyl-tRNA Synthetase Has Glutamyl-tRNA Synthetase Activity.

The aminoacyl-tRNA synthetases (aaRSs) are well established as the translators of the genetic code, because their products, the aminoacyl-tRNAs, read codons to translate messenger RNAs into proteins. Consequently, deleterious errors by the aaRSs can be transferred into the proteome via misacylated tRNAs. Nevertheless, many microorganisms use an indirect pathway to produce Asn-tRNAAsn via Asp-tRNAAsn. This intermediate is produced by a non-discriminating aspartyl-tRNA synthetase (ND-AspRS) that has retained its ability to also generate Asp-tRNAAsp. Here we report the discovery that ND-AspRS and its discriminating counterpart, AspRS, are also capable of specifically producing Glu-tRNAGlu, without producing misacylated tRNAs like Glu-tRNAAsn, Glu-tRNAAsp, or Asp-tRNAGlu, thus maintaining the fidelity of the genetic code. Consequently, bacterial AspRSs have glutamyl-tRNA synthetase-like activity that does not contaminate the proteome via amino acid misincorporation.

DNA methyltransferase homologue TRDMT1 in Plasmodium falciparum specifically methylates endogenous aspartic acid tRNA.

In eukaryotes, cytosine methylation regulates diverse biological processes such as gene expression, development and maintenance of genomic integrity. However, cytosine methylation and its functions in pathogenic apicomplexan protozoans remain enigmatic. To address this, here we investigated the presence of cytosine methylation in the nucleic acids of the protozoan Plasmodium falciparum. Interestingly, P. falciparum has TRDMT1, a conserved homologue of DNA methyltransferase DNMT2. However, we found that TRDMT1 did not methylate DNA, in vitro. We demonstrate that TRDMT1 methylates cytosine in the endogenous aspartic acid tRNA of P. falciparum. Through RNA bisulfite sequencing, we mapped the position of 5-methyl cytosine in aspartic acid tRNA and found methylation only at C38 position. P. falciparum proteome has significantly higher aspartic acid content and a higher proportion of proteins with poly aspartic acid repeats than other apicomplexan pathogenic protozoans. Proteins with such repeats are functionally important, with significant roles in host-pathogen interactions. Therefore, TRDMT1 mediated C38 methylation of aspartic acid tRNA might play a critical role by translational regulation of important proteins and modulate the pathogenicity of the malarial parasite.

Crystal structure of the N-terminal anticodon-binding domain of the nondiscriminating aspartyl-tRNA synthetase from Helicobacter pylori.

The N-terminal anticodon-binding domain of the nondiscriminating aspartyl-tRNA synthetase (ND-AspRS) plays a crucial role in the recognition of both tRNAAsp and tRNAAsn. Here, the first X-ray crystal structure of the N-terminal domain of this enzyme (ND-AspRS1-104) from the human-pathogenic bacterium Helicobacter pylori is reported at 2.0 Šresolution. The apo form of H. pylori ND-AspRS1-104 shares high structural similarity with the N-terminal anticodon-binding domains of the discriminating aspartyl-tRNA synthetase (D-AspRS) from Escherichia coli and ND-AspRS from Pseudomonas aeruginosa, allowing recognition elements to be proposed for tRNAAsp and tRNAAsn. It is proposed that a long loop (Arg77-Lys90) in this H. pylori domain influences its relaxed tRNA specificity, such that it is classified as nondiscriminating. A structural comparison between D-AspRS from E. coli and ND-AspRS from P. aeruginosa suggests that turns E and F (78GAGL81 and 83NPKL86) in H. pylori ND-AspRS play a crucial role in anticodon recognition. Accordingly, the conserved Pro84 in turn F facilitates the recognition of the anticodons of tRNAAsp (34GUC36) and tRNAAsn (34GUU36). The absence of the amide H atom allows both C and U bases to be accommodated in the tRNA-recognition site.

Osmium tetroxide as a probe of RNA structure.

Structured RNAs have a central role in cellular function. The capability of structured RNAs to adopt fixed architectural structures or undergo dynamic conformational changes contributes to their diverse role in the regulation of gene expression. Although numerous biophysical and biochemical tools have been developed to study structured RNAs, there is a continuing need for the development of new methods for the investigation of RNA structures, especially methods that allow RNA structure to be studied in solution close to its native cellular conditions. Here we use osmium tetroxide (OsO4) as a chemical probe of RNA structure. In this method, we have used fluorescence-based sequencing technologies to detect OsO4 modified RNA. We characterized the requirements for OsO4 modification of RNA by investigating three known structured RNAs: the M-box, glycine riboswitch RNAs, and tRNAasp Our results show that OsO4 predominantly modifies RNA at uracils that are conformationally exposed on the surface of the RNA. We also show that changes in OsO4 reactivity at flexible positions in the RNA correlate with ligand-driven conformational changes in the RNA structure. Osmium tetroxide modification of RNA will provide insights into the structural features of RNAs that are relevant to their underlying biological functions.

Somatic cancer mutations in the DNMT2 tRNA methyltransferase alter its catalytic properties.

Methylation of tRNA is an important post-transcriptional modification and aberrations in tRNA modification has been implicated in cancer. The DNMT2 protein methylates C38 of tRNA-Asp and it has a role in cellular physiology and stress response and its expression levels are altered in cancer tissues. Here we studied whether DNMT2 somatic mutations found in cancer tissues affect the activity of the enzyme. We have generated 13 DNMT2 variants and purified the corresponding proteins. All proteins were properly folded as determined by circular dichroism spectroscopy. We tested their RNA methylation activity using in vitro generated tRNA-Asp. One of the mutations (E63K) caused a twofold increase in activity, while two of them led to a strong (over fourfold) decrease in activity (G155S and L257V). Two additional mutant proteins were almost inactive (R371H and G155V). The strong effect of some of the somatic cancer mutations on DNMT2 activity suggests that these mutations have a functional role in tumorigenesis.

Dynamics of RNA modification by a multi-site-specific tRNA methyltransferase.

In most organisms, the widely conserved 1-methyl-adenosine58 (m1A58) tRNA modification is catalyzed by an S-adenosyl-L-methionine (SAM)-dependent, site-specific enzyme TrmI. In archaea, TrmI also methylates the adjacent adenine 57, m1A57 being an obligatory intermediate of 1-methyl-inosine57 formation. To study this multi-site specificity, we used three oligoribonucleotide substrates of Pyrococcus abyssi TrmI (PabTrmI) containing a fluorescent 2-aminopurine (2-AP) at the two target positions and followed the RNA binding kinetics and methylation reactions by stopped-flow and mass spectrometry. PabTrmI did not modify 2-AP but methylated the adjacent target adenine. 2-AP seriously impaired the methylation of A57 but not A58, confirming that PabTrmI methylates efficiently the first adenine of the A57A58A59 sequence. PabTrmI binding provoked a rapid increase of fluorescence, attributed to base unstacking in the environment of 2-AP. Then, a slow decrease was observed only with 2-AP at position 57 and SAM, suggesting that m1A58 formation triggers RNA release. A model of the protein-tRNA complex shows both target adenines in proximity of SAM and emphasizes no major tRNA conformational change except base flipping during the reaction. The solvent accessibility of the SAM pocket is not affected by the tRNA, thereby enabling S-adenosyl-L-homocysteine to be replaced by SAM without prior release of monomethylated tRNA.

Relaxed tRNA specificity of the Staphylococcus aureus aspartyl-tRNA synthetase enables RNA-dependent asparagine biosynthesis.

The human pathogen Staphylococcus aureus is an asparagine prototroph despite its genome not encoding an asparagine synthetase. S. aureus does use an asparaginyl-tRNA synthetase (AsnRS) to directly ligate asparagine to tRNA(Asn). The S. aureus genome also codes for one aspartyl-tRNA synthetase (AspRS). Here we demonstrate the lone S. aureus aspartyl-tRNA synthetase has relaxed tRNA specificity and can be used with the amidotransferase GatCAB to synthesize asparagine on tRNA(Asn). S. aureus thus encodes both the direct and indirect routes for Asn-tRNA(Asn) formation while encoding only one aspartyl-tRNA synthetase. The presence of the indirect pathway explains how S. aureus synthesizes asparagine without either asparagine synthetase.

The Dnmt2 RNA methyltransferase homolog of Geobacter sulfurreducens specifically methylates tRNA-Glu.

Dnmt2 enzymes are conserved in eukaryotes, where they methylate C38 of tRNA-Asp with high activity. Here, the activity of one of the very few prokaryotic Dnmt2 homologs from Geobacter species (GsDnmt2) was investigated. GsDnmt2 was observed to methylate tRNA-Asp from flies and mice. Unexpectedly, it had only a weak activity toward its matching Geobacter tRNA-Asp, but methylated Geobacter tRNA-Glu with good activity. In agreement with this result, we show that tRNA-Glu is methylated in Geobacter while the methylation is absent in tRNA-Asp. The activities of Dnmt2 enzymes from Homo sapiens, Drosophila melanogaster, Schizosaccharomyces pombe and Dictyostelium discoideum for methylation of the Geobacter tRNA-Asp and tRNA-Glu were determined showing that all these Dnmt2s preferentially methylate tRNA-Asp. Hence, the GsDnmt2 enzyme has a swapped transfer ribonucleic acid (tRNA) specificity. By comparing the different tRNAs, a characteristic sequence pattern was identified in the variable loop of all preferred tRNA substrates. An exchange of two nucleotides in the variable loop of murine tRNA-Asp converted it to the corresponding variable loop of tRNA-Glu and led to a strong reduction of GsDnmt2 activity. Interestingly, the same loss of activity was observed with human DNMT2, indicating that the variable loop functions as a specificity determinant in tRNA recognition of Dnmt2 enzymes.

Target recognition, RNA methylation activity and transcriptional regulation of the Dictyostelium discoideum Dnmt2-homologue (DnmA).

Although the DNA methyltransferase 2 family is highly conserved during evolution and recent reports suggested a dual specificity with stronger activity on transfer RNA (tRNA) than DNA substrates, the biological function is still obscure. We show that the Dictyostelium discoideum Dnmt2-homologue DnmA is an active tRNA methyltransferase that modifies C38 in tRNA(Asp(GUC)) in vitro and in vivo. By an ultraviolet-crosslinking and immunoprecipitation approach, we identified further DnmA targets. This revealed specific tRNA fragments bound by the enzyme and identified tRNA(Glu(CUC/UUC)) and tRNA(Gly(GCC)) as new but weaker substrates for both human Dnmt2 and DnmA in vitro but apparently not in vivo. Dnmt2 enzymes form transient covalent complexes with their substrates. The dynamics of complex formation and complex resolution reflect methylation efficiency in vitro. Quantitative PCR analyses revealed alterations in dnmA expression during development, cell cycle and in response to temperature stress. However, dnmA expression only partially correlated with tRNA methylation in vivo. Strikingly, dnmA expression in the laboratory strain AX2 was significantly lower than in the NC4 parent strain. As expression levels and binding of DnmA to a target in vivo are apparently not necessarily accompanied by methylation, we propose an additional biological function of DnmA apart from methylation.

Positioning of CCA-arms of the A- and the P-tRNAs towards the 28S rRNA in the human ribosome.

Nucleotides of 28S rRNA involved in binding of the human 80S ribosome with acceptor ends of the A site and the P site tRNAs were determined using two complementary approaches, namely, cross-linking with application of tRNA(Asp) analogues substituted with 4-thiouridine in position 75 or 76 and hydroxyl radical footprinting with the use of the full sized tRNA and the tRNA deprived of the 3'-terminal trinucleotide CCA. In general, these 28S rRNA nucleotides are located in ribosomal regions homologous to the A, P and E sites of the prokaryotic 50S subunit. However, none of the approaches used discovered interactions of the apex of the large rRNA helix 80 with the acceptor end of the P site tRNA typical with prokaryotic ribosomes. Application of the results obtained to available atomic models of 50S and 60S subunits led us to a conclusion that the A site tRNA is actually present in both A/A and A/P states and the P site tRNA in the P/P and P/E states. Thus, the present study gives a biochemical confirmation of the data on the structure and dynamics of the mammalian ribosomal pretranslocation complex obtained with application of cryo-electron microscopy and single-molecule FRET [Budkevich et al., 2011]. Moreover, in our study, particular sets of 28S rRNA nucleotides involved in oscillations of tRNAs CCA-termini between their alternative locations in the mammalian 80S ribosome are revealed.

Pmt1, a Dnmt2 homolog in Schizosaccharomyces pombe, mediates tRNA methylation in response to nutrient signaling.

The fission yeast Schizosaccharomyces pombe carries a cytosine 5-methyltransferase homolog of the Dnmt2 family (termed pombe methyltransferase 1, Pmt1), but contains no detectable DNA methylation. Here, we found that Pmt1, like other Dnmt2 homologs, has in vitro methylation activity on cytosine 38 of tRNA(Asp) and, to a lesser extent, of tRNA(Glu), despite the fact that it contains a non-consensus residue in catalytic motif IV as compared with its homologs. In vivo tRNA methylation also required Pmt1. Unexpectedly, however, its in vivo activity showed a strong dependence on the nutritional status of the cell because Pmt1-dependent tRNA methylation was induced in cells grown in the presence of peptone or with glutamate as a nitrogen source. Furthermore, this induction required the serine/threonine kinase Sck2, but not the kinases Sck1, Pka1 or Tor1 and was independent of glucose signaling. Taken together, this work reveals a novel connection between nutrient signaling and tRNA methylation that thus may link tRNA methylation to processes downstream of nutrient signaling like ribosome biogenesis and translation initiation.

Insights into the hyperthermostability and unusual region-specificity of archaeal Pyrococcus abyssi tRNA m1A57/58 methyltransferase.

The S-adenosyl-L-methionine dependent methylation of adenine 58 in the T-loop of tRNAs is essential for cell growth in yeast or for adaptation to high temperatures in thermophilic organisms. In contrast to bacterial and eukaryotic tRNA m(1)A58 methyltransferases that are site-specific, the homologous archaeal enzyme from Pyrococcus abyssi catalyzes the formation of m(1)A also at the adjacent position 57, m(1)A57 being a precursor of 1-methylinosine. We report here the crystal structure of P. abyssi tRNA m(1)A57/58 methyltransferase ((Pab)TrmI), in complex with S-adenosyl-L-methionine or S-adenosyl-L-homocysteine in three different space groups. The fold of the monomer and the tetrameric architecture are similar to those of the bacterial enzymes. However, the inter-monomer contacts exhibit unique features. In particular, four disulfide bonds contribute to the hyperthermostability of the archaeal enzyme since their mutation lowers the melting temperature by 16.5°C. His78 in conserved motif X, which is present only in TrmIs from the Thermococcocales order, lies near the active site and displays two alternative conformations. Mutagenesis indicates His78 is important for catalytic efficiency of (Pab)TrmI. When A59 is absent in tRNA(Asp), only A57 is modified. Identification of the methylated positions in tRNAAsp by mass spectrometry confirms that (Pab)TrmI methylates the first adenine of an AA sequence.

Tertiary network in mammalian mitochondrial tRNAAsp revealed by solution probing and phylogeny.

Primary and secondary structures of mammalian mitochondrial (mt) tRNAs are divergent from canonical tRNA structures due to highly skewed nucleotide content and large size variability of D- and T-loops. The nonconservation of nucleotides involved in the expected network of tertiary interactions calls into question the rules governing a functional L-shaped three-dimensional (3D) structure. Here, we report the solution structure of human mt-tRNA(Asp) in its native post-transcriptionally modified form and as an in vitro transcript. Probing performed with nuclease S1, ribonuclease V1, dimethylsulfate, diethylpyrocarbonate and lead, revealed several secondary structures for the in vitro transcribed mt-tRNA(Asp) including predominantly the cloverleaf. On the contrary, the native tRNA(Asp) folds into a single cloverleaf structure, highlighting the contribution of the four newly identified post-transcriptional modifications to correct folding. Reactivities of nucleotides and phosphodiester bonds in the native tRNA favor existence of a full set of six classical tertiary interactions between the D-domain and the variable region, forming the core of the 3D structure. Reactivities of D- and T-loop nucleotides support an absence of interactions between these domains. According to multiple sequence alignments and search for conservation of Leontis-Westhof interactions, the tertiary network core building rules apply to all tRNA(Asp) from mammalian mitochondria.

Pathology-related mutation A7526G (A9G) helps in the understanding of the 3D structural core of human mitochondrial tRNA(Asp).

More than 130 mutations in human mitochondrial tRNA (mt-tRNA) genes have been correlated with a variety of neurodegenerative and neuromuscular disorders. Their molecular impacts are of mosaic type, affecting various stages of tRNA biogenesis, structure, and/or functions in mt-translation. Knowledge of mammalian mt-tRNA structures per se remains scarce however. Primary and secondary structures deviate from classical tRNAs, while rules for three-dimensional (3D) folding are almost unknown. Here, we take advantage of a myopathy-related mutation A7526G (A9G) in mt-tRNA(Asp) to investigate both the primary molecular impact underlying the pathology and the role of nucleotide 9 in the network of 3D tertiary interactions. Experimental evidence is presented for existence of a 9-12-23 triple in human mt-tRNA(Asp) with a strongly conserved interaction scheme in mammalian mt-tRNAs. Mutation A7526G disrupts the triple interaction and in turn reduces aspartylation efficiency.

Native-like RNA tertiary structures using a sequence-encoded cleavage agent and refinement by discrete molecular dynamics.

The difficulty of analyzing higher order RNA structure, especially for folding intermediates and for RNAs whose functions require domains that are conformationally flexible, emphasizes the need for new approaches for modeling RNA tertiary structure accurately. Here, we report a concise approach that makes use of facile RNA structure probing experiments that are then interpreted using a computational algorithm, carefully tailored to optimize both the resolution and refinement speed for the resulting structures, without requiring user intervention. The RNA secondary structure is first established using SHAPE chemistry. We then use a sequence-directed cleavage agent, which can be placed arbitrarily in many helical motifs, to obtain high quality inter-residue distances. We interpret this in-solution chemical information using a fast, coarse grained, discrete molecular dynamics engine in which each RNA nucleotide is represented by pseudoatoms for the phosphate, ribose, and nucleobase groups. By this approach, we refine base paired positions in yeast tRNA(Asp) to 4 A rmsd without any preexisting information or assumptions about secondary or tertiary structures. This blended experimental and computational approach has the potential to yield native-like models for the diverse universe of functionally important RNAs whose structures cannot be characterized by conventional structural methods.

Sites of 18S rRNA contacting mRNA 3' and 5' of the P site codon in human ribosome: a cross-linking study with mRNAs carrying 4-thiouridines at specific positions.

Long synthetic mRNAs were used to study the positioning of the E site codon, the 2nd and 3rd nucleotides of the A site bound codon and a nucleotide 3' of this codon with respect to the 18S rRNA in the human 80S ribosome. The mRNAs contained a GAC triplet coding for Asp and a single 4-thiouridine residue (s(4)U) upstream or downstream of the GAC codon. In the presence of tRNA(Asp), the GAC codon of the mRNAs was targeted to the ribosomal P site thus placing s(4)U in one of the following positions -3, -2, -1, +5, +6 or +7 with respect to the first nucleotide of the P site bound codon. It was found that mRNAs that bore s(4)U in positions +5 to +7 cross-linked to the 18S rRNA nucleotides C1696, C1698 and 1820-1825, the distribution of cross-links among these targets depending on the position of s(4)U. Cross-links of mRNAs containing s(4)U in positions -3 to -1 were found in the region 1699-1704 of the 18S rRNA. In the absence of tRNA, all mRNAs cross-linked only to C1696 and C1698. Absence of the cross-linked nucleotides C1696 and C1698 in the case of mRNAs containing s(4)U in positions -3 to -1 confirmed that tRNA(Asp) actually phased the mRNA on the ribosome.

RNA cytosine methylation analysis by bisulfite sequencing.

Covalent modifications of nucleic acids play an important role in regulating their functions. Among these modifications, (cytosine-5) DNA methylation is best known for its role in the epigenetic regulation of gene expression. Post-transcriptional RNA modification is a characteristic feature of noncoding RNAs, and has been described for rRNAs, tRNAs and miRNAs. (Cytosine-5) RNA methylation has been detected in stable and long-lived RNA molecules, but its function is still unclear, mainly due to technical limitations. In order to facilitate the analysis of RNA methylation patterns we have established a protocol for the chemical deamination of cytosines in RNA, followed by PCR-based amplification of cDNA and DNA sequencing. Using tRNAs and rRNAs as examples we show that cytosine methylation can be reproducibly and quantitatively detected by bisulfite sequencing. The combination of this method with deep sequencing allowed the analysis of a large number of RNA molecules. These results establish a versatile method for the identification and characterization of RNA methylation patterns, which will be useful for defining the biological function of RNA methylation.

[Artificial ribonucleases: quantitative analysis of the structure-activity relationship and new insight into the strategy of design of highly efficient RNase mimetics].

The dependence of hydrolytic activity of artificial ribonucleases toward an HIV-I RNA fragment, a 21-mer oligonucleotide, and tRNA Asp on the structure of the RNase mimetic was analyzed. The quantitative structure-activity relationship (QSAR task) was determined by the method of simplex representation of the molecular structure where the amounts of four-atom fragments (simplexes) of fixed structure, symmetry, and chirality served as descriptors. Not only the types of atoms participating in simplexes but also their physicochemical properties (e.g., partial charges, lipophilicities, etc.) were taken into account. This allowed the estimation of the relative role of various factors affecting the interaction of molecules under study with the corresponding biological target. The 2D QSAR models obtained by the method of projection to latent structures have quite satisfactory statistical indices (R2 = 0.82-0.96; Q2 = 0.73-0.89), which help predict the activities of new compounds. The electrostatic properties of ribonuclease atoms were shown to contribute significantly to the manifestation of the hydrolytic activity of ribonucleases in the case of the 21-mer oligonucleotide and tRNA. In addition, the structural fragments that most greatly contribute to the alteration of the hydrolytic activity of RNases were identified. The models obtained were used for the virtual screening and molecular design of new highly efficient RNase mimetics.