S-Geranyl-2-thiouridine wobble nucleosides of bacterial tRNAs; chemical and enzymatic synthesis of S-geranylated-RNAs and their physicochemical characterization.

Recently, highly lipophilic S-geranylated derivatives of 5-methylaminomethyl-2-thiouridine (mnm5geS2U) and 5-carboxymethylaminomethyl-2-thiouridine (cmnm5geS2U) were found at the first (wobble) anticodon position in bacterial tRNAs specific for Lys, Glu and Gln. The function and cellular biogenesis of these unique tRNAs remain poorly understood. Here, we present one direct and two post-synthetic chemical routes for preparing model geS2U-RNAs. Our experimental data demonstrate that geS2U-RNAs are more lipophilic than their parent S2U-RNAs as well as non-modified U-RNAs. Thermodynamic studies revealed that the S-geranyl-2-thiouridine-containing RNA has higher affinity toward complementary RNA strand with G opposite the modified unit than with A. Recombinant tRNA selenouridine synthase (SelU) exhibits sulfur-specific geranylation activity toward model S2U-RNA, which is composed of the anticodon-stem-loop (ASL) from the human tRNALys3 sequence. In addition, the presence of magnesium ions is required to achieve appreciable geranylation efficiencies.

Creating small transcription activating RNAs.

We expanded the mechanistic capability of small RNAs by creating an entirely synthetic mode of regulation: small transcription activating RNAs (STARs). Using two strategies, we engineered synthetic STAR regulators to disrupt the formation of an intrinsic transcription terminator placed upstream of a gene in Escherichia coli. This resulted in a group of four highly orthogonal STARs that had up to 94-fold activation. By systematically modifying sequence features of this group, we derived design principles for STAR function, which we then used to forward engineer a STAR that targets a terminator found in the Escherichia coli genome. Finally, we showed that STARs could be combined in tandem to create previously unattainable RNA-only transcriptional logic gates. STARs provide a new mechanism of regulation that will expand our ability to use small RNAs to construct synthetic gene networks that precisely control gene expression.

RNA synthetic biology inspired from bacteria: construction of transcription attenuators under antisense regulation.

Among all biopolymers, ribonucleic acids or RNA have unique functional versatility, which led to the early suggestion that RNA alone (or a closely related biopolymer) might have once sustained a primitive form of life based on a single type of biopolymer. This has been supported by the demonstration of processive RNA-based replication and the discovery of 'riboswitches' or RNA switches, which directly sense their metabolic environment. In this paper, we further explore the plausibility of this 'RNA world' scenario and show, through synthetic molecular design guided by advanced RNA simulations, that RNA can also perform elementary regulation tasks on its own. We demonstrate that RNA synthetic regulatory modules directly inspired from bacterial transcription attenuators can efficiently activate or repress the expression of other RNA by merely controlling their folding paths 'on the fly' during transcription through simple RNA-RNA antisense interaction. Factors, such as NTP concentration and RNA synthesis rate, affecting the efficiency of this kinetic regulation mechanism are also studied and discussed in the light of evolutionary constraints. Overall, this suggests that direct coupling among synthesis, folding and regulation of RNAs may have enabled the early emergence of autonomous RNA-based regulation networks in absence of both DNA and protein partners.

Probing mRNA structure and sRNA-mRNA interactions in bacteria using enzymes and lead(II).

Enzymatic probing and lead(II)-induced cleavages have been developed to study the secondary structure of RNA molecules either free or engaged in complex with different ligands. Using a combination of probes with different specificities (unpaired vs. paired regions), it is possible to get information on the accessibility of each nucleotide, on the binding site of a ligand (noncoding RNAs, protein, metabolites), and on RNA conformational changes that accompanied ligand binding or environmental conditions (temperature, pH, ions, etc.). The detection of the cleavages can be conducted by two different ways, which are chosen according to the length of the studied RNA. The first method uses end-labeled RNA molecules and the second one involves primer extension by reverse transcriptase. We provide here an experimental procedure that was designed to map the structure of mRNA and mRNA-sRNA interaction in vitro.

Evaluation of bacterial RNase P RNA as a drug target.

RNA has gained increasing importance as a therapeutic target. However, so far mRNAs rather than stable cellular RNAs have been considered in such studies. In bacteria, the tRNA-processing enzyme RNase P has a catalytic RNA subunit. Fundamental differences in structure and function between bacterial and eukaryotic RNase P, and its indispensability for cell viability make the bacterial enzyme an attractive drug target candidate. Herein we describe two approaches utilized to evaluate whether the catalytic RNA subunit of bacterial RNase P is amenable to inactivation by antisense-based strategies. In the first approach, we rationally designed RNA hairpin oligonucleotides targeted at the tRNA 3'-CCA binding site (P15 loop region) of bacterial RNase P RNA by attempting to include principles derived from the natural CopA-CopT antisense system. Substantial inactivation of RNase P RNA was observed for Type A RNase P RNA (such as that in Escherichia coli) but not for Type B (as in Mycoplasma hyopneumoniae). Moreover, only an RNA oligonucleotide (Eco 3') complementary to the CCA binding site and its 3' flanking sequences was shown to be an efficient inhibitor. Mutation of Eco 3' and analysis of other natural RNase P RNAs with sequence deviations in the P15 loop region showed that inhibition is due to interaction of Eco 3' with this region and occurs in a highly sequence-specific manner. A DNA version of Eco 3' was a less potent inhibitor. The potential of Eco 3' to form an initial kissing complex with the P15 loop did not prove advantageous. In a second approach, we tested a set of oligonucleotides against E. coli RNase P RNA which were designed by algorithms developed for the selection of suitable mRNA targets. This approach identified the P10/11-J11/12 region of bacterial RNase P RNA as another accessible region. In conclusion, both the P15 loop and P10/11-J11/12 regions of Type A RNase P RNAs seem to be promising antisense target sites since they are easily accessible and sufficiently interspersed with nonhelical sequence elements, and oligonucleotide binding directly interferes with substrate docking to these two regions.

Antisense inhibition of Escherichia coli RNase P RNA: mechanistic aspects.

The ribonucleoprotein enzyme RNase P catalyzes endonucleolytic 5'-maturation of tRNA primary transcripts in all domains of life. The indispensability of RNase P for bacterial cell growth and the large differences in structure and function between bacterial and eukaryotic RNase P enzymes comply with the basic requirements for a bacterial enzyme to be suitable as a potential novel drug target. We have identified RNA oligonucleotides that start to show an inhibitory effect on bacterial RNase P RNAs of the structural type A (for example, the Escherichia coli or Klebsiella pneumoniae enzymes) at subnanomolar concentrations in our in vitro precursor tRNA (ptRNA) processing assay. These oligonucleotides are directed against the so-called P15 loop region of RNase P RNA known to interact with the 3'-CCA portion of ptRNA substrates. Lead probing experiments demonstrate that a complementary RNA or DNA 14-mer fully invades the P15 loop region and thereby disrupts local structure in the catalytic core of RNase P RNA. Binding of the RNA 14-mer is essentially irreversible because of a very low dissociation rate. The association rate of this oligonucleotide is on the order of 10(4) M(-1) s(-1) and is thus comparable to those of many other artificial antisense oligonucleotides. The remarkable inhibition efficacy is attributable to the dual effect of direct interference with substrate binding to the RNase P RNA active site and induction of misfolding of the catalytic core of RNase P RNA. Based on our findings, the P15 loop region of bacterial RNase P RNAs of the structural type A can be considered the "Achilles' heel" of the ribozyme and therefore represents a promising target for combatting multiresistant bacterial pathogens.

Synthesis of helix 69 of Escherichia coli 23S rRNA containing its natural modified nucleosides, m(3)Psi and Psi.

The synthesis of 3-methylpseudouridine (m(3)Psi) phosphoramidite, 5'-O-[benzhydryloxybis(trimethylsilyloxy)silyl]-2'-O-[bis(2-acetoxyethoxy)methyl]-3-methylpseudouridine-3'-(methyl-N,N-diisopropyl)phosphoramidite, is reported. Selective pivaloyloxymethyl protection of the Psi N1 followed by methylation at N3 was used to generate the naturally occurring pseudouridine analogue. The m(3)Psi phosphoramidite was used in combination with pseudouridine (Psi) and standard base phosphoramidites to synthesize a 19-nucleotide RNA representing helix 69 of Escherichia coli 23S ribosomal RNA (rRNA) (residues 1906-1924), containing a single m(3)Psi at position 1915 and two Psi's at positions 1911 and 1917. Our synthesis of the fully modified helix 69 RNA demonstrates the ability to make milligram quantities of RNA that can be used for further high-resolution structure studies. Site-selective introduction of the methyl group at the N3 position of pseudouridine at position 1915 causes a slight increase in the thermodynamic stability of the RNA hairpin relative to pseudouridine; RNAs containing either uridine or 3-methyluridine at position 1915 have similar stability. One-dimensional imino proton NMR and circular dichroism spectra of the modified RNAs reveal that the methyl group does not cause any substantial changes in the RNA hairpin structure.

Stereospecificity of aminoglycoside-ribosomal interactions.

Aminoglycoside antibiotics bind to the A-site decoding region of bacterial rRNA causing mistranslation and/or premature message termination. Aminoglycoside binding to A-site RNA decoding region constructs is established here to be only weakly stereospecific. Mirror-image prokaryotic A-site decoding region constructs were prepared in the natural D-series and the enantiomeric L-series and tested for binding to a series of aminoglycosides. In general, aminoglycosides bind to the D-series decoding region constructs with 2-3-fold higher affinities than they bind to the enantiomeric L-series. Moreover, L-neamine, the enantiomer of naturally occurring D-neamine, was prepared and shown to bind approximately 2-fold more weakly than D-neamine to the natural series decoding region construct, a result consistent with weakly stereospecific binding. The binding of naturally occurring D-neamine and its synthetic L-enantiomer was further evaluated with respect to binding to prokaryotic and eukaryotic ribosomes. Here, weak stereospecifcity was again observed with L-neamine being the more potent binder by a factor of approximately 2. However, on a functional level, unnatural L-neamine proved to inhibit in vitro translation with significantly lower potency (approximately 5-fold) than D-neamine. In addition, both L- and D-neamine are bacteriocidal toward Gram-(-) bacteria. L-Neamine inhibits the growth of E. coli and P. aeruginosa with 8- and 3-fold higher MIC than D-neamine. Interestingly, L-neamine also inhibits the growth of aminoglycoside-resistant E. coli, which expresses a kinase able to phosphorylate and detoxify aminoglycosides of the D-series. These observations suggest that mirror-image aminoglycosides may avoid certain forms of enzyme-mediated resistance.

Synthesis and characterization of the native anticodon domain of E. coli TRNA(Lys): simultaneous incorporation of modified nucleosides mnm(5)s(2)U, t(6)A, and pseudouridine using phosphoramidite chemistry.

The anticodon domain of E. coli tRNA(Lys) contains the hypermodified nucleosides mnm(5)s(2)U and t(6)A at positions 34 and 37, respectively, along with a more common psi at position 39. The combination of these three nucleotides represents one of the most extensively modified RNA domains in nature. 2-Cyanoethyl diisopropylphosphoramidites of the hypermodified nucleosides mnm(5)s(2)U and t(6)A were each synthesized with protecting groups suitable for automated RNA oligonucleotide synthesis. The 17 nucleotide anticodon stem-loop of E. coli tRNA(Lys) was then assembled from these synthons using phosphoramidite coupling chemistry. Coupling efficiencies for the two hypermodified nucleosides and for pseudouridine phosphoramidite were all greater than 98%. A mild deprotection scheme was developed to accommodate the highly functionalized RNA. High coupling yields, mild deprotection, and efficient HPLC purification allowed us to obtain 1. 8 mg of purified RNA from a 1 micromol scale RNA synthesis. Our efficient synthetic protocol will allow for biophysical investigation of this rather unique tRNA species wherein nucleoside modification has been shown to play a role in codon-anticodon recognition, tRNA aminoacyl synthetase recognition, and programmed ribosomal frameshifting. The human analogue, tRNA(Lys,3), is the specific tRNA primer for HIV-1 reverse transcriptase and has a similar modification pattern.

Identification of 2'-hydroxyl groups required for interaction of a tRNA anticodon stem-loop region with the ribosome.

Synthetic RNA stem loops corresponding to positions 28-42 in the anticodon region of tRNA(Phe) bind efficiently in an mRNA-dependent manner to ribosomes, whereas those made from DNA do not. In order to identify the positions where ribose is required, the anticodon stem-loop region of tRNA(Phe) (Escherichia coli) was synthesized chemically using a mixture of 2'-hydroxyl- and 2'-deoxynucleotide phosphoramidites. Oligonucleotides whose ribose composition allowed binding were retained selectively on nitrocellulose filters via binding to 30S ribosomal subunits. The binding-competent oligonucleotides were submitted to partial alkaline hydrolysis to identify the positions that were enriched for ribose. Quantification revealed a strong preference for a 2'-hydroxyl group at position U33. This was shown directly by the 50-fold lower binding affinity of a stem loop containing a single deoxyribose at position U33. Similarly, defective binding of the corresponding U33-2'-O-methyl-substituted stem-loop RNA suggests that absence of the 2'-hydroxyl group, rather than an altered sugar pucker, is responsible. Stem-loop oligoribonucleotides from different tRNAs with U33-deoxy substitutions showed similar, although quantitatively different effects, suggesting that intramolecular rather than tRNA-ribosome interactions are affected. Because the 2'-hydroxyl group of U33 was shown to be a major determinant of the U-turn of the anticodon loop in the crystal structure of tRNA(Phe) in yeast, our finding might indicate that the U-turn conformation in the anticodon loop is required and/or maintained when the tRNA is bound to the ribosomal P site.

The synthesis and functional evaluation of RNA and DNA polymers having the sequence of Escherichia coli tRNA(fMet).

Stepwise, solid-phase chemical synthesis has provided long RNA and DNA polymers related to the sequence of Escherichia coli tRNA(fMet). The 34-ribonucleotide oligomer corresponding to the sequence of the 5'-half tRNA molecule has been synthesized and then characterized by gel purification, terminal nucleotide determinations and sequence analysis. This 34-nucleotide oligomer serves as an acceptor in the RNA-ligase-catalyzed reaction with a phosphorylated 43-ribonucleotide oligomer corresponding to the sequence of the 3'-half molecule of tRNA(fMet). The DNA molecule having the sequence of tRNA(fMet) is a 76-deoxyribonucleotide oligomer with a 3'-terminal riboadenosine residue and all U residues replaced by T. These polymers have been compared with an oligodeoxyribonucleotide lacking all 2'-hydroxyl groups except for the 3'-terminal 2'-OH, an oligoribonucleotide lacking modified nucleosides and E. coli tRNA(fMet). The all-RNA 77-nucleotide oligomer can be aminoacylated by E. coli methionyl-tRNA synthetase preparation from E. coli with methionine and threonylated in the A37 position using a yeast extract. In agreement with work by Khan and Roe using tDNA(Phe) and tDNA(Lys), the rA77-DNA(fMet) can be aminoacylated, and preliminary evidence suggests that it can be threonylated to a small extent. Kinetic data support the notion that aminoacylation of tRNA(fMet) does not depend on the presence of 2'-hydroxyl groups with the exception of that in the 3'-terminal nucleotide.

Studies towards the synthesis of the fluorescent bases of phenylalanine transfer ribonucleic acids: synthesis of 7-methylwye isolated from extremely thermophilic archaebacteria.

Acid- or base-catalyzed acylation of 1-benzylwye (7) provided the 7-substituted derivatives 9, 10, and 11 in poor yields. Although the reactions of lithiated 7 with electrophiles gave the 2-substituted derivatives 14, 15, 17, 20, 21, and 22, lithiation of 1-benzyl-7-bromo-2-chlorowye (23) followed by treatment with Me2CHCH2CHO (13) successfully introduced a side chain at the 7-position to afford 1-benzyl-2-chloro-7-(1-hydroxy-3-methylbutyl)wye (24). Cyclization of 1-benzyl-3-methylguanine (5) with 3-bromo-2-butanone followed by catalytic hydrogenolysis afforded 7-methylwye (2b), the hypermodified base isolated from archaebacterial transfer ribonucleic acids. A more efficient route for the synthesis of 2b has been developed via a series of reactions: the Vilsmeier-Haack reaction of 7, reduction with NaBH4, and catalytic hydrogenolysis over Pd-C.

Labile protecting groups in tRNA synthesis.

The use of labile protecting groups for the protection of the exocyclic amino function of adenine, guanine and cytosine has two main advantages in RNA synthesis. Final deprotection in concentrated aqueous ammonia takes place in milder conditions which are more compatible with the sensitivity of oligoribonucleotides towards alkali-conditions. The introduction of fragile bases such as certain modified bases encountered in the primary structure of t-RNA is feasible. The chemical synthesis of RNA fragments constituting the primary structure of B. subtilis f-methionine t-RNA is described.