Pyranosyl-RNA ('p-RNA'): base-pairing selectivity and potential to replicate

A review and discussion, with 42 refs. Base pairing in p-RNA (b-D-ribopyranosyl-(4'->2')-oligonucleotides) is not only stronger than in DNA and RNA, but also more selective in the sense that it is strictly confined to the Watson-Crick mode. Homopurine sequences (tested up to decamers) exist as single strands under conditions where they undergo reverse-Hoogsteen self-pairing in homo-DNA or Hoogsteen self-pairing in DNA. This exceptional pairing selectivity is rationalized as hinging on 2 structural features of p-RNA: the large inclination between backbone axis and base-pair axes in p-RNA duplexes, and the higher rigidity of the p-RNA backbone compared with RNA, DNA, and homo-DNA. The most important consequence of the pairing selectivity refers to the potential of p-RNA to replicate. Replicative copying of sequence information by nonenzymic template-controlled ligation is not hampered by self-pairing of guanine-rich templates, as it is known to be the case in the RNA series. Two replicative cycles are demonstrated in which G-rich p-RNA-octamer templates induce sequence-selective ligation of tetramer-2'-phosphate derivs. to complementary C-rich octamer sequences, and in which the latter, with comparable efficiency, induce corresponding ligation reactions back to the original G-rich octamers. Ligation is most satisfactorily achieved after pre-activation of the 2'-phosphate groups as 2',3'-cyclophosphate derivs.; in this version, the process does not proceed as oligocondensation, but as a genuine oligomerization. This is of considerable promise for the search for potentially natural conditions under which homochiral p-RNA strands might self-assemble and self-replicate. [on SciFinder (R)]

The use of RNA interference to increase PEI-mediated transient gene expression in mammalian cells

Martin Bertschinger

DNA uptake by polyethylenimine (PEI)-mediated transfection was investigated in Chinese hamster ovary (CHO DG44) cells. Rapid DNA uptake was observed with the maximum occurring during the first 45-60 min after addition of PEI-DNA complexes to cells. With longer incubation times, the aggregation of PEI-DNA particles reduced the rate of DNA uptake. At early times after transfection, the kinetics of DNA uptake were constant, suggesting that the limiting factor to PEI transfection was the rate of endocytosis. The most efficient transfection with PEI was observed at a density of 2 x 106 cells/ml and at a PEI:DNA ratio of 2:1 (w/w). DNA uptake at higher PEI:DNA ratios was also efficient, but the toxicity of PEI decreased the recombinant protein yield. The optimal PEI:DNA ratio was found to be related to the number of cells in the culture. Once in the cell, PEI:DNA particles must be disassembled to allow transcription of the recombinant gene. Here their disassembly was investigated using a simple in vitro assay. The particles were formed with either a linear (25 kDa or JetPEITM) or a branched PEI (2, 25, or 750 kDa) and then mixed with varying amounts of a competitor (RNA, DNA, or heparin). The amount of plasmid DNA released from particles was determined by agarose gel electrophoresis or spectroscopy. The stability of the particles to changes in pH, osmolarity, and the PEI:DNA ratio was also determined. Either the presence of heparin or high salt concentrations (1-2 M) yielded complete particle disassembly for all PEIs tested. The addition of RNA to particles formed with linear PEIs or branched 2 kDa PEI resulted in the rapid release of all the plasmid DNA, even at an RNA concentration of 50 µg/ml. However, the presence of RNA at concentrations up to 400 µg/ml induced only partial disassembly of particles formed with branched PEIs of 25 or 750 kDa. In the presence of competitor DNA, slow particle disassembly was observed with particles made with linear 25 kDa PEI, JetPEI, and branched 2 kDa PEI, but DNA release was not seen for particles made with branched PEIs of higher molecular weight. The presence of BSA only resulted in partial disassembly of PEI-DNA particles, and DNA release was not observed after the exposure of particles to acidic pH as low as 3. For all the PEIs tested, their stability increased as the PEI:DNA ratio increased. It was concluded from these studies that branched PEIs have a higher affinity for DNA than linear PEIs and that the intracellular disassembly of PEI-DNA particles may involve interactions between PEI and cellular RNA. In the second part of the work, RNA interference (RNAi) was used to enhance transient gene expression by knockdown of two different mRNAs, the E1A mRNA in human embryonic kidney 293 EBNA (HEK293E) cells and the ube2i mRNA in CHO DG44 cells and HEK293 cells. HEK293 cells are transformed with the adenovirus E1A and E1B genes. Because the E1A proteins function as transcriptional activators or repressors, they may have a positive or negative effect on transient transgene expression in this cell line. Suspension cultures of HEK293 cells were co-transfected with a reporter plasmid expressing the GFP gene and a plasmid expressing a short hairpin RNA (shRNA) targeting the E1A mRNAs for degradation by RNAi. The presence of the shRNA in HEK293E cells reduced the steady state level of E1A mRNA up to 75% and increased transient GFP expression from either the elongation factor-1α (EF-1α) promoter or the human cytomegalovirus (HCMV) immediate early promoter up to 3-fold. E1A mRNA depletion also resulted in a 2-fold increase in transient expression of a recombinant IgG in suspension cultures when the IgG light and heavy chain genes were driven by the EF-1α promoter. These results demonstrated that E1A has a negative effect on transient gene expression in HEK293E cells. Efficient RNAi-mediated depletion of ube2i mRNA in CHO DG44 cells was accomplished by co-expression of 2 shRNAs targeting two distinct sides in the ube2i mRNA, whereas knockdown with only one of the shRNAs at a time was insufficient for reducing ube2i mRNA levels. The reduction of the ube2i mRNA level in CHO DG44 cells was shown to enhance transient GFP expression 2-fold and IgG expression 2.5-fold. For these experiments, the GFP gene was under the control of the CMV IE promoter and the IgG light and heavy chain genes were under the control of the human EF-1α promoter. Thus, the enhancement of transient gene expression resulting from ube2i mRNA depletion was not promoter-dependent. These results demonstrated that sumoylation has a negative effect on transient gene expression in CHO DG44 and HEK293E cells.

EPFL2006

New methods for the investigation of RNA refolding by NMR spectroscopy

Philipp Wenter

A detailed characterization of the time scales and mechanisms by which RNA molecules change their folding structure upon binding of metabolites or during catalysis is important for the understanding of the different biological functions of RNA. The folding from an unfolded, denaturated state has been extensively studied, establishing hierarchical pathways, in which fast formation of secondary structural elements precedes the folding of tertiary elements. In contrast, only a few examples of time-resolved RNA refolding in the native state have been reported so far. Specifically, NMR spectroscopy has not yet been employed for the investigation of RNA refolding despite its intrinsic sensibility to dynamic structural changes. In the project presented here, new tools for the identification of RNA secondary structures and the quantification of RNA refolding processes by NMR spectroscopy have been developed and employed. Preparation of 15N-labeled phosphoramidites and their sequence-selective introduction into RNA sequences allowed a straightforward identification of base pairs and imino protons by HNN-COSY and 15N HSQC experiments, respectively. Synthesis and introduction of NPE-protected uridines and guanosines into bistable RNA sequences provided a powerful method for the photoinduced preparation of selected RNA folds far from equilibrium. When introduced into bistable RNA sequences they allow to disrupt selected base pairs, thereby destabilizing the associated secondary structures. As a consequence, one out of usually two coexisting fold could be prepared selectively in its caged form. Upon photolysis, the native sequence was released under physiological conditions with completely retained, preselected secondary structure arrangement. Refolding into the thermodynamic equilibrium was subsequently followed by real-time imino proton NMR spectroscopy, providing quantitative, time-resolved and structural information for the refolding processes of three bistable RNA sequences and a Mg2+-induced secondary and tertiary structure refolding. Depending on their topology, these RNA sequences refold either via a dissociative mechanism in which the disruption of existing base pairs precedes the formation of new ones or via an associative mechanism in which new base pairs are formed from initially unpaired sequence regions simultaneously to the detachement of existing base pairs. In the transition state approximately half of the base pairs are disrupted. The investigation of a bistable RNA sequence designed to undergo a topologically favored refolding processes was carried out by exchange sensitive NMR experiments. The introduction of sequence specific 15N-labeles into RNA sequences allowed the implementation of exchange sensitive 1D and 2D 1H/15N-heteronuclear NMR methods which were designed to map very slow exchange. We found that a 34mer RNA sequence exhibits two folds which exchange on the observable time scale (τobs = T1{15N}

EPFL2006

Preparation of aminoacylated tRNAs and analogues

The ribosome-mediated, site-specific incorporation of unnatural amino acids into proteins is a powerful tool for creating protein analogues with specific steric, chemical, electronic or spectroscopic properties, in vitro and in vivo. The required aminoacylated tRNA analogues are prepared semisynthetically, by ribozyme-catalyzed amino acid transfer from short aminoacylated RNA sequences, from complementary PNA amino acid thioesters or by modified aminoacyl-tRNA synthetases. The common semisynthetic preparation of aminoacylated tRNA analogues is based on the ligation of aminoacylated dimers with a truncated tRNA, produced by run-off transcription. However, the presence of overtranscripts and the lability of the amino acid/nucleotide ester bond represent a severe limitation in terms of purity and efficiency of this ligation reaction. In this context, we first have optimized the in vitro RNA transcription reaction to obtain a straightforward access to homogeneous truncated tRNAs. We found that addition of 3.5 equivalents of GMP results not only in the selective formation of transcripts with 5'-monophosphate termini, but also in the formation of less overtranscripts without affecting the yield. Moreover, we have investigated the purification of in vitro transcription reaction mixtures by anion exchange HPLC, resulting in a simple and efficient isolation/purification protocol (Chapter 2.1). We have prepared tRNAs aminoacylated with L-lysine -BODIPY and L-lysine-fluorescein conjugates, respectively, by efficient blunt-end ligation of a corresponding, protected and stabilized aminoacylated dimer with truncated tRNAs. The resulting protected and stabilized aminoacylated tRNAs could be characterized by ESI-MS. The presence of two photolabile groups at the 3'-terminal 2'-OH group and on the α-amino group of the amino acid allowed an efficient preparation and purification of the aminoacylated tRNA analogues and to store them for an unlimited time at -20° (Chapter 2.2). Although RNA transcription is an easy method to prepare truncated tRNAs, this method suffers from severe limitations to introduce modified nucleotides into tRNAs. To overcome this limitation, we have optimized their preparation from synthetic RNA fragments by T4-DNA ligase mediated ligation reactions. An amber suppressor tRNA derived from the E.coli tRNAPhe was used as model system and a ligation from three RNA fragments was investigated. Specifically, the design of the templates and the reaction conditions were evaluated and optimized. We found that 2'-OMeRNA templates or DNA/2'-OMeRNA hybrid templates could lead to a significantly better conversion than the commonly employed DNA templates. Under optimized conditions, the full tRNA could be assembled with an efficiency of 65% (Chapter 2.3). Finally within the context of a new concept for tRNA aminoacylation, we have prepared a biotinyl-lysine tRNA containing a 3'-terminal 2'-deoxy-2'-thioadenosine. The new concept was developed in our group by S. Porcher and M. Meyyappan, who found that such RNA analogues react efficiently, spontaneously and site-specifically with weakly activated amino acid thioesters in buffered aqueous solutions and under a wide range of conditions (Chapter 2.4). Some efforts towards the development of translation assays are described. Unfortunately, the corresponding experiments were not successful (Chapter 2.5).

EPFL2006

The use of RNA interference to increase PEI-mediated transient gene expression in mammalian cells

Martin Bertschinger

EPFL2006

Preparation of aminoacylated tRNAs and analogues

EPFL2006

New methods for the investigation of RNA refolding by NMR spectroscopy

Philipp Wenter

EPFL2006