Two-terpyridine.Cu(II) complexes-containing antisense systems for rapid and highly site-specific RNA cleavage.

In an approach toward artificial ribonucleases, novel RNA cleaving systems were constructed that contained two terpyridine.Cu(II) residues. The first antisense system used tandem Cu(II) complex--2'-O-methyloligonucleotide 5'- and 3'-conjugates to cleave an RNA substrate. The second system, which will be described in a future paper, contained two contiguous Cu(II) complex residues at an internal site of a 2'-O-methyloligonucleotide. We found that the first system rapidly cleaved RNA with high site-specificity. Based on these results, we expect the second system to also show efficient RNA cleavage.     

Analysis of mRNA recognition by human thymidylate synthase

Expression of hTS (human thymidylate synthase), a key enzyme in thymidine biosynthesis, is regulated on the translational level through a feedback mechanism that is rarely found in eukaryotes. At low substrate concentrations, the ligand-free enzyme binds to its own mRNA and stabilizes a hairpin structure that sequesters the start codon. When in complex with dUMP (2′-deoxyuridine-5′-monophosphate) and a THF (tetrahydrofolate) cofactor, the enzyme adopts a conformation that is unable to bind and repress expression of mRNA. Here, we have used a combination of X-ray crystallography, RNA mutagenesis and site-specific cross-linking studies to investigate the molecular recognition of TS mRNA by the hTS enzyme. The interacting mRNA region was narrowed to the start codon and immediately flanking sequences. In the hTS enzyme, a helix–loop–helix domain on the protein surface was identified as the putative RNA-binding site.     

Characterization of a specific interaction between Escherichia coli thymidylate synthase and Escherichia coli thymidylate synthase mRNA.

Previous studies have shown that human TS mRNA translation is controlled by a negative autoregulatory mechanism. In this study, an RNA electrophoretic gel mobility shift assay confirmed a direct interaction between Escherichia coli (E.coli) TS protein and its own E.coli TS mRNA. Two cis-acting sequences in the E.coli TS mRNA protein-coding region were identified, with one site corresponding to nucleotides 207-460 and the second site corresponding to nucleotides 461-807. Each of these mRNA sequences bind TS with a relative affinity similar to that of the full-length E.coli TS mRNA sequence (IC50 = 1 nM). A third binding site was identified, corresponding to nucleotides 808-1015, although its relative affinity for TS (IC50 = 5.1 nM) was lower than that of the other two cis-acting elements. E.coli TS proteins with mutations in amino acids located within the nucleotide-binding region retained the ability to bind RNA while proteins with mutations at either the nucleotide active site cysteine (C146S) or at amino acids located within the folate-binding region were unable to bind TS mRNA. These studies suggest that the regions on E.coli TS defined by the folate-binding site and/or critical cysteine sulfhydryl groups may represent important RNA binding domains. Further evidence is presented which demonstrates that the direct interaction with TS results in in vitro repression of E.coli TS mRNA translation.     

The identification of thymidylate synthase peptide domains located in the interface region that bind thymidylate synthase mRNA

Thymidylate synthase (TS) is a critical chemotherapeutic target and intracellular levels of TS are an important determinant of sensitivity to TS inhibitors. Translational autoregulation represents one cellular mechanism for controlling the level of expression of TS. This mechanism involves the binding of TS protein to its own messenger RNA (mRNA), thus, repressing translational efficiency. The presence of excess substrate or inhibitors of TS leads to derepression of protein binding to mRNA, resulting in increased translational efficiency and ultimately increased levels of TS protein. TS protein has been shown to bind to two distinct areas on its mRNA. The goal of the present work is to define the TS domains responsible for this interaction. Using a separate series of overlapping 17-mer peptides spanning the length of both the human and Escherichia coli TS sequences, we have identified six potential domains located in the interface region of the TS protein that bind TS mRNA. The identified domains that bind TS mRNA include three concordant regions in both the human and E. coli peptide series. Five of the six binding peptides contain at least one invariant arginine residue, which has been shown to be critical in other well-defined protein-RNA interactions. These data suggest that the identified highly conserved protein domains, which occur at the homodimeric interface of TS, represent potential participating sites for binding of TS protein to its mRNA.     

Photomodulating RNA cleavage using photolabile circular antisense oligodeoxynucleotides

Caged antisense oligodeoxynucleotides (asODNs) are synthesized by linking two ends of linear oligodeoxynucleotides using a photocleavable linker. Two of them (H30 and H40) have hairpin-like structures which show a large difference in thermal stability (ΔT_m = 17.5°C and 11.6°C) comparing to uncaged ones. The other three (C20, C30 and C40) without stable secondary structures have the middle 20 deoxynucleotides complementary to 40-mer RNA. All caged asODNs have restricted opening which provides control over RNA/asODN interaction. RNase H assay results showed that 40-mer RNA digestion could be photo-modulated 2- to 3-fold upon light-activation with H30, H40, C30 and C40, while with C20, RNA digestion was almost not detectable; however, photo-activation triggered >20-fold increase of RNA digestion. And gel shift assays showed that it needed >0.04 μM H40 and 0.5 μM H30 to completely bind 0.02 μM 40-mer RNA, and for C40 and C30, it needed >0.2 μM and 0.5 μM for 0.02 μM 40-mer RNA binding. However, even 4 μM C20 was not able to fully bind the same concentration of 40-mer RNA. By simple adjustment of ring size of caged asODNs, we could successfully photoregulate their hybridization with mRNA and target RNA hydrolysis by RNase H with light activation.     

Fluorescence spectroscopic and (19)f NMR studies of human thymidylate synthase with its cognate RNA

In order to investigate the interaction between hTS protein and its cognate mRNA, a 29nt fragment of TS mRNA was synthesized. This region has been suggested as a putative stem-loop involved in translational autoregulation. The melting temperature of the 29ntRNA was 65 degrees C, suggesting that this region does indeed form a stem-loop. Fluorescence spectroscopy was used to monitor the RNA: hTS protein interaction [dissociation constant (K(d)) 3.9 +/- 0.8 nM; stoichiometry of binding 1dimeric hTS: 1RNA]. When hTS was titrated against FdUMP, this gave the expected stoichiometry of 1dimeric hTS: 1.7 FdUMP but in the presence of the 29ntRNA, the stoichiometry of binding changed to 1dimeric hTS: 1RNA: 1FdUMP. Experiments using methotrexate (MTX) gave a stoichiometry of 1dimeric hTS: 1MTX and in the presence of 29ntRNA, the stoichiometry was unchanged. (19)F-NMR spectra of human TS: FdUMP complexes were found to be strikingly similar to analogous NMR spectra of complexes formed by L.casei TS and mouse TS. In the presence of FdUMP, spectra exhibited two additional resonances (-1.50 ppm and -34.4 ppm). The resonance at -1.50 ppm represents non-covalently bound FdUMP, the peak at -34.4 ppm represents covalently bound FdUMP. The addition of methotrexate to the binary TS-FdUMP complex caused a displacement of the internal equilibrium, with only the covalently-bound form seen, and with a slightly disturbed (19)F chemical shift (-36.5 ppm). Similar results were found when MTX was replaced by folinic or folic acid. The addition of 29ntRNA caused no changes to the (19)F spectra of either the binary or ternary complexes.     

The role of thymidylate synthase as an RNA binding protein

Thymidylate synthase plays a central role in the biosynthesis of thymidylate, an essential precursor for DNA biosynthesis. In addition to its role in catalysis and cellular metabolism, it is now appreciated that thymidylate synthase functons as an RNA binding protein. Specifically, thymidylate synthase binds with high affinity to its own mRNA, resulting in translational repression. An extensive series of experiments has been performed to elucidate the molecular elements underlying the interaction between thymidylate synthase and its own mRNA. In addition to characterization of the underlying cis- and trans-acting elements, recent studies have shown that thymidylate synthase has the capacity to bind specifically to other cellular RNA species. While the biological significance of these other RNA/thymidylate synthase interactions remains to be defined, this work suggests a potential role for TS in coordinately regulating several critical aspects of cellular metabolism.     

Single-stranded antisense siRNAs guide target RNA cleavage in RNAi

Small interfering RNAs (siRNAs) are the mediators of mRNA degradation in the process of RNA interference (RNAi). Here, we describe a human biochemical system that recapitulates siRNA-mediated target RNA degradation. By using affinity-tagged siRNAs, we demonstrate that a single-stranded siRNA resides in the RNA-induced silencing complex (RISC) together with eIF2C1 and/or eIF2C2 (human GERp95) Argonaute proteins. RISC is rapidly formed in HeLa cell cytoplasmic extract supplemented with 21 nt siRNA duplexes, but also by adding single-stranded antisense RNAs, which range in size between 19 and 29 nucleotides. Single-stranded antisense siRNAs are also effectively silencing genes in HeLa cells, especially when 5'-phosphorylated, and expand the repertoire of RNA reagents suitable for gene targeting.     

Rapid RNA cleavage using an antisense system with two terpyridine.Cu(II) complexes

An effective approach to promote sequence-specific RNA cleavage by an antisense 2'-O-methyloligonucleotide with a terpyridine.Cu(II) complex attached at the 5'-end was developed. We have synthesized a Cu(II) complex 3'-conjugate, which when used in a tandem fashion, greatly enhanced the RNA cleavage efficiency.     

Specific binding of Hoechst 33258 to site 1 thymidylate synthase mRNA

The translational initiator codon in thymidylate synthetase (TS) mRNA is located in a stem–loop structure with a CC bubble. TS is an important target for anticancer drugs. Aminoglycoside antibiotics have been shown to specifically bind to TS mRNA site 1 constructs and, furthermore, specific binding requires the non-duplex CC bubble region. It is shown here that DNA intercalating agents and DNA minor groove-binding drugs also bind to a TS mRNA site 1 construct. This binding is competitive with aminoglycosides, suggesting that the binding sites overlap. Hoechst 33258 binds with a dissociation constant of 60 nM, a value significantly lower than the ~1 µM values found for aminoglycosides. Footprinting and direct binding studies show that the CC bubble is important for binding of the Hoechst compound. However, the exact structure of the bubble is unimportant. Interestingly, mutations in regions adjacent to the bulge also affect binding. These studies point to the important role of non-duplex RNA structures in binding of the DNA minor groove binder Hoechst 33258.     

Determination of optimal sites of antisense oligonucleotide cleavage within TNFalpha mRNA

Antisense oligonucleotides provide a powerful tool in order to determine the consequences of the reduced expression of a selected target gene and may include target validation and therapeutic applications. Methods of predicting optimum antisense sites are not always effective. We have compared the efficacy of antisense oligonucleotides, which were selected in vitro using random combinatorial oligonucleotide libraries of differing length and complexity, upon putative target sites within TNFα mRNA. The relationship of specific target site accessibility and oligonucleotide efficacy with respect to these parameters proved to be complex. Modification of the length of the recognition sequence of the oligonucleotide library illustrated that independent target sites demonstrated a preference for antisense oligonucleotides of a defined and independent optimal length. The efficacy of antisense oligonucleotide sequences selected in vitro paralleled that observed in phorbol 12-myristate 13-acetate (PMA)-activated U937 cells. The application of methylphosphonate:phosphodiester chimaeric oligonucleotides to U937 cells reduced mRNA levels to up to 19.8% that of the untreated cell population. This approach provides a predictive means to profile any mRNA of known sequence with respect to the identification and optimisation of sites accessible to antisense oligonucleotide activity.     

V(D)J recombination: site-specific cleavage and repair

V(D)J recombination is a site-specific gene rearrangement process that contributes to the diversity of antigen receptor repertoires. Two lymphoid-specific proteins, RAG1 and RAG2, initiate this process at two recombination signal sequences. Due to the recent development of an in vitro assay for V(D)J cleavage, the mechanism of cleavage has been elucidated clearly. The RAG complex recognizes a recombination signal sequence, makes a nick at the border between signal and coding sequence, and carries out a transesterification reaction, resulting in the production of a hairpin structure at the coding sequence and DNA double-strand breaks at the signal ends. RAG1 possesses the active site of the V(D)J recombinase although RAG2 is essential for signal binding and cleavage. After DNA cleavage by the RAG complex, the broken DNA ends are rejoined by the coordinated action of DNA double-strand break repair proteins as well as the RAG complex. The junctional variability resulting from imprecise joining of the coding sequences contributes additional diversity to the antigen receptors.     

Highly efficient site-directed RNA cleavage by imidazole-containing conjugates of antisense oligonucleotides

A method has been suggested for the synthesis of conjugates of oligodeoxyribonucleotides with chemical constructs mimicking ribonuclease A active center for directed fragmentation of RNA. The method is based on the sequential addition of linker group, 9-(methylamino)anthracene, to 5' or 3' terminal phosphate of oligonucleotide and then imidazole-containing construct by cycloaddition reaction. The conjugates of oligonucleotides complementary to regions 44-61 (2B-R) and 60-76 (1C-R) of yeast phenylalanine tRNA demonstrated ability to cleave tRNA(Phe) under physiological conditions preferably at the sole phosphodiester bond (C63-A64 for 2B-R and C56-G57 for 1C-R, respectively). The half-time of tRNA(Phe) hydrolysis in the presence of 2B-R conjugate was 30 min at 2B-R concentration of 10 microM and several minutes at conjugate concentration of 50 microM.     

Identification of in vivo target RNA sequences bound by thymidylate synthase.

We developed an immunoprecipitation-RNA-random PCR (rPCR) method to isolate cellular RNA sequences that bind to the folate-dependent enzyme thymidylate synthase (TS). Using this approach, nine different cellular RNAs that formed a ribonucleoprotein (RNP) complex with thymidylate synthase (TS) in human colon cancer cells were identified. RNA binding experiments revealed that seven of these RNAs bound TS with relatively high affinity (IC50 values ranging from 1.5 to 6 nM). One of the RNAs was shown to encode the interferon (IFN)-induced 15 kDa protein. Western immunoblot analyses demonstrated that the level of IFN-induced 15 kDa protein was significantly decreased in human colon cancer H630-R10 cells compared with parent H630 cells. While the level of IFN-induced 15 kDa mRNA expression was the same in parent and TS-overexpressing cell lines, the level of IFN-induced 15 kDa RNA bound to TS in the form of a RNP complex was markedly higher in H630-R10 cells relative to parent H630 cells. These studies begin to define a number of cellular target RNA sequences with which TS interacts and suggest that these TS protein-cellular RNA interactions may have a biological role.     

Remote control of mRNA cleavage by a small RNA

Comment on: Prévost K, et al. Genes Dev 2011; 25:385-96.     

Improved efficiency of site-specific copper(II) ion-catalysed protein cleavage effected by mutagenesis of cleavage site

The peptide sequence (N)DKTH(C) was previously investigated as a site for efficient, specific cleavage of a fusion protein by cupric ions using a humanized gamma1 Fab' as a model protein. Here we show that conservative mutations to three of the residues in the introduced cleavage site resulted in cleavage sites that were significantly improved. They were cleaved more efficiently by Cu(2+), such that cleavage reactions could be shorter, of lower pH or at a lower temperature. Some were even found to be measurably cleaved by Ni(2+). Use of these new cleavage sequences along with cupric ions may provide a more rapid and less harsh method for cost-effective, large-scale proteolytic cleavage of fusion proteins and peptides.