No abstract

A 58-mer L-RNA ligand that binds to naturally occurring D-adenosine with a dissociation constant of 1.7 microM in solution was identified from a combinatorial library employing mirror-design. The corresponding D-RNA ligand shows identical binding affinity to L-adenosine. Reciprocal chiral specificity was also evident from ligand discrimination; the binding affinity of the L-RNA ligand for D-adenosine was 9000-fold greater than its affinity for L-adenosine and vice versa. While the D-RNA ligand was rapidly degraded in human serum, the L-RNA ligand displayed an extraordinary stability. This indicates the potential application of specifically designed L-RNA ligands as stable monoclonal antibody analogues and the development of highly stable L-ribozymes.

A systematic mutagenesis study of the "10-23" DNA enzyme was performed to analyze the sequence requirements of its catalytic domain. Therefore, each of the 15 core nucleotides was substituted separately by the remaining three naturally occurring nucleotides. Changes at the borders of the catalytic domain led to a dramatic loss of enzymatic activity, whereas several nucleotides in between could be exchanged without severe effects. Thymidine at position 8 had the lowest degree of conservation and its substitution by any of the other three nucleotides caused only a minor loss of activity. In addition to the standard nucleotides (adenosine, guanosine, thymidine, or cytidine) modified nucleotides were used to gain further information about the role of individual functional groups. Again, thymidine at position 8 as well as some other nucleotides could be substituted by inosine without severe effects on the catalytic activity. For two positions, additional experiments with 2-aminopurine and deoxypurine, respectively, were performed to obtain information about the specific role of functional groups. In addition to sequence-function relationships of the DNA enzyme, this study provides information about suitable sites to introduce modified nucleotides for further functional studies or for internal stabilization of the DNA enzyme against endonucleolytic attack.

To study the cleavage mechanism ofbacterial Nase P RNA, we have synthesized precursor tRNA substrates carrying a single Rp-or RNase P is an essential structure-specific endoribonuclease that generates the mature 5' ends of tRNAs. In vitro, RNA subunits of bacterial RNase P enzymes were shown to be catalytically active in the absence of the protein subunit (1). Processing of precursor tRNAs (ptRNAs) by RNase P is an essentially irreversible reaction yielding 3'-OH and 5'-phosphate termini. A solvent hydroxide is thought to act as the nucleophile in an SN2 in-line displacement mechanism (2, 3).To gain a deeper insight into the cleavage mechanism by Escherichia coli RNase P RNA, we have synthesized ptRNA substrates carrying a single Rp-or Sp-phosphorothioate modification at the RNase P cleavage site. The diastereomeric substrates were analyzed for gel-resolvable binding to RNase P RNA and were studied in single turnover experiments in the presence of different divalent metal ions, such as Mg2+, Mn2+, and Cd2+. The following results were obtained: (i) the Spdiastereomer moderately affected ptRNA ground state binding, while the Rp-diastereomer had no effect; (ii) cleavage by RNase P RNA involves direct metal ion coordination to the pro-Rp oxygen; (iii) there is no specific role for Mg2+ at the pro-Sp oxygen; and (iv) cleavage of the Rp-diastereomeric substrate has a lower cooperative dependence (nH = 1.8) upon[Cd2+] than cleavage of the unmodified substrate upon [Mg2+] (nH = 3.3). Implications for the unique RNase P RNA cleavage mechanism are discussed in the context of previously proposed mechanistic models (2-4). MATERIALS AND METHODS Separation and Identification of Diastereomeric 13-Mers.Oligoribonucleotides of identical sequence (13-mers), either unmodified or carrying a single phosphorothiote modification (5'-CCCUUUCsGCGGGA), were prepared by solid-phase synthesis essentially as described (5, 6). Oligoribonucleotides were purified by reversed-phase HPLC on an ODS C18 Beckman Ultrasphere column at 45°C (5). The material of the peak containing the two diastereomeric 13-mers was vacuumdried, and the Rp-and Sp-diastereomers were separated by a second reversed-phase run at 4°C. The assignment of configuration of the two diastereomeric 13-mers was accomplished by digestion with the stereoselective enzymes snake venom phosphodiesterase I or nuclease P1 essentially as described (6). Phosphorothioate-specific iodine hydrolysis was performed essentially as described recently (7).Assembly of the ptRNAGIY. Chemically synthesized RNA oligomers were phosphorylated at their 5' termini by T4 polynucleotide kinase; T7 transcripts were synthesized in the presence of excess 5'-GMP to obtain 5'-monophosphates (7). Modified or unmodified 13-mers, a second 11-nt RNA oligonucleotide (5'-GUAGCUCAGUC-3', obtained either by chemical RNA synthesis or T7 transcription), and the 3'-portion of the tRNA (obtained by T7 transcription, starting at G+ 18; see Fig. 1) were annealed to a bridging DNA oligonucleotide (complementary to...

To characterize protein-DNA interactions involved in the initiation of conjugative transfer replication, we isolated and sequenced the transfer origins (ori7) of the promiscuous IncP plasmids RP4 and R751. The central initiating event at the transfer origin of a conjugative plasmid is the cleavage at a unique site (nic) of the strand to be transferred to a recipient cell. This process can be triggered after the assembly of "relaxosomes" (plasmid DNA-protein relaxation complexes), requiring plasmid-encoded gene products. We analyzed the nicking reaction for plasmid RP4 and demonstrated that one of the plasmid strands is specifically cleaved within onT. The fully functional oriT of RP4 represents an intergenic DNA region of -350 base pairs. Dissection of oriT revealed that a portion carrying nic and symmetric sequence repeats determines oriT specificity. This part of oriT is contiguous to a region that is essential for efficient mobilization of oriT plasmids. In addition, oriT contains potential promoter sites allowing divergent transcription of two operons flanking oriT. We overproduced gene products and, from analyzing the products of dermed deletion mutants, deduced the gene arrangements. Formation of RP4 relaxosomes is likely to depend on the presence of at least two plasmid-encoded components, which act in trans. Corresponding genes map on one side of onT. Purification of the traJ product revealed it to be an 11-kDa polypeptide that binds to oriT DNA in vitro. The protein recognizes the part of oriT that is responsible for oriT specificity. While most of the transfer functions, including the matingpair formation system, can be utilized by both plasmids, the interaction at the transfer origin of tra gene products is plasmid specific (6). We used this observation to map the genes encoding oriT-specific functions within the regions immediately flanking oriT. Furthermore, an electrophoretic assay was developed to analyze rapidly the nicking reaction; the assay allowed us to locate genes required for relaxosome nicking within the region encoding oriT-specific functions. Expression-vector cloning of fragments carrying oriTspecific functions facilitated the analysis ofgene organization and the overproduction of gene products. One of these proteins, the traJ gene product, specifically binds to the oriT region in vitro, thus suggesting an important role of the protein in triggering the initial events of transfer DNA replication.Conjugation is the process that allows efficient gene transfer from one bacterial cell to another through plasmid-encoded functions. Conjugative plasmids of the IncP group are of particular interest because they are capable of mediating efficient DNA transfer between virtually any Gram-negative species (1). This promiscuity is important because of its role in the spread of antibiotic resistance and its application to gene manipulation in widely different bacteria.Conjugative transfer requires both a cis-acting site, the origin of transfer (oriT), and a number of trans-acting functions t...

The high affinity and selectivity of nucleic acid ligands have clearly demonstrated that RNA can be targeted to a variety of molecules. In practice, however, the use of unmodified aptamers is impeded by the low stability of RNA in biological fluids. Here we describe the mirror-design of a stable 38-mer L-oligoribonucleotide ligand that binds to L-arginine. This L-RNA ligand was also able to bind to a short peptide containing the basic region of the human immunodeficiency virus type-1 Tat-protein. The L-RNA ligand displayed the expected stability in human serum. These findings may contribute to the identification of novel diagnostics and pharmaceuticals.

No abstract

‘Locked nucleic acids’ (LNAs) are known to introduce enhanced bio- and thermostability into natural nucleic acids rendering them powerful tools for diagnostic and therapeutic applications. We present the 1.9 Å X-ray structure of an ‘all LNA’ duplex containing exclusively modified β-d-2′-O-4′C-methylene ribofuranose nucleotides. The helix illustrates a new type of nucleic acid geometry that contributes to the understanding of the enhanced thermostability of LNA duplexes. A notable decrease of several local and overall helical parameters like twist, roll and propeller twist influence the structure of the LNA helix and result in a widening of the major groove, a decrease in helical winding and an enlarged helical pitch. A detailed structural comparison to the previously solved RNA crystal structure with the corresponding base pair sequence underlines the differences in conformation. The surrounding water network of the RNA and the LNA helix shows a similar hydration pattern.