Theoretical design of antisense RNA structures substantially improves annealing kinetics and efficacy in human cells.

The success of antisense therapeutics is not predictable despite their widespread use in biotechnology and molecular medicine. The relationship between RNA structure and biological effectiveness is largely not understood; however, antisense RNA-mediated effects in vivo seem to be related to annealing kinetics in vitro. This study suggests that terminal unpaired nucleotides and overall flexibility of antisense RNA directed against the human immunodeficiency virus type 1 (HIV-1) are related to fast RNA-RNA annealing in vitro as well as to strong inhibition of virus replication in human cells. Annealing rate constants of computer-selected antisense RNA species approach the values for natural antisense RNA in the order of 10(6) M-1s-1. When considering the unfavorable stability in cellular extracts of antisense RNA species that were found to anneal fast in vitro, an antisense effect against HIV-1 in human cells was observed that was 10- to 10,000-fold stronger than that measured for species predicted to anneal slowly. A computer-supported structural design of antisense RNA can serve as a platform to determine RNA-RNA association in vitro and biological effectiveness in living cells.

In vitro selection supports the view of a kinetic control of antisense RNA-mediated inhibition of gene expression in mammalian cells.

In principle, the steady-state concentrations of biomolecules in complex systems can be far from the thermodynamic equilibrium concentrations of individual processes. This means that, in addition to thermodynamics, reaction kinetics may play an important role. This view is not fully reflected in combinatorial studies in biochemistry that focus on the selection of stably interacting molecules reflected by high equilibrium constants. For kinetically controlled processes in vivo, forward or backward reaction rates are critical but not necessarily an equilibrium state. Here we have studied the control of antisense RNA-mediated gene suppression in human cells on a general basis and in a way that excludes individual structure-specific influences. The complete antisense sequence space against the chloramphenicol acetyltransferase gene (cat) was generated and a kinetic selection technique was established to enrich for fast annealing antisense species. Selected sub-populations showed successively faster annealing which was related to increased inhibition of cat gene expression in HeLa cells, providing strong evidence for the view that the suppression of gene expression by antisense RNA is controlled kinetically regardless of specific RNA structures.

RNA accessibility prediction: a theoretical approach is consistent with experimental studies in cell extracts.

The use of antisense oligodeoxyribonucleotides (ODN) or ribozymes to specifically suppress gene expression is simple in concept and relies on efficient binding of the antisense strand to the target RNA. Although the identification of target sites accessible to base pairing is gradually being overcome by different techniques, it remains a major problem in the antisense and ribozyme approaches. In this study we have investigated the potential of a recent experimental and theoretical approach to predict the local accessibility of murine DNA-methyltransferase (MTase) mRNA in a comparative way. The accessibility of the native target RNA was probed with antisense ODN in cellular extracts. The results strongly correlated with the theoretically predicted target accessibility. This work suggests an effective two-step procedure for predicting RNA accessibility: first, computer-aided selection of ODN binding sites defined by an accessibility score followed by a more detailed experimental procedure to derive information about target accessibility at the single nucleotide level.

Theoretical design of antisense genes with statistically increased efficacy.

Endogenous expression of antisense RNA represents one major way of applying antisense nucleic acids. To express antisense RNA intracellularly, recombinant antisense genes have to be designed and introduced into cells where the target RNA is encountered. Efficient annealing between the antisense RNA and the target RNA is crucial for efficacy and is strongly influenced by RNA structure. Here we extend structural rules for the design of in vitro transcribed antisense RNAs to the design of recombinant antisense genes. Intracellularly expressed antisense RNA transcripts contain a central antisense portion and additional flanking vector-derived sequences. A computer algorithm was generated to compose large sets of antisense genes, to calculate secondary structures of the transcribed sequences and to select for favorable structures of antisense RNA in terms of annealing and efficacy. The biological test system to measure efficiency of antisense genes was human immunodeficiency virus type 1 (HIV-1) replication in 293T cells. When considering the lower intracellular steady-state levels of favorably structured endogenous transcripts, an antisense effect against HIV-1 replication was observed that was up to 60-fold stronger than that measured for predicted unfavorable species. The computational selection was successful for antisense portions of 300 nt but not 100 nt in length. This theoretical design of antisense genes supports their improved application under time- and labor-saving conditions.

A theoretical approach to select effective antisense oligodeoxyribonucleotides at high statistical probability.

Up to now, out of approximately 20 antisense oligodeoxyribonucleotides (as ODN) selected and tested against a given target gene, only one species shows substantial suppression of target gene expression. In part, this seems to be related to the general assumption that the structures of local target sequences or antisense nucleic acids are unfavorable for efficient annealing. Experimental approaches to find effective as ODN are extremely expensive when including a large number of antisense species and when considering their moderate success. Here, we make use of a systematic alignment of computer-predicted secondary structures of local sequence stretches of the target RNA and of semi-empirical rules to identify favorable local target sequences and, hence, to design more effective as ODN. The intercellular adhesion molecule 1 (ICAM-1) gene was chosen as a target because it had been shown earlier to be sensitive to antisense-mediated gene suppression. By applying the protocol described here, 10 ICAM-1-directed as ODN species were found that showed substantially improved inhibition of target gene expression in the endothelial cell line ECV304 when compared with the most effective published as ODN. Further, 17 out of 34 antisense species (50%) selected on the theoretical basis described here showed significant (>50%) inhibition of ICAM-1 expression in mammalian cells.

The potential of ribozymes as antiviral agents.

Ribozymes are promising tools for the specific inhibition of viral gene expression and replication. They represent one of the most attractive developments of antisense nucleic acids, which have been shown in the past few years to act as antiviral agents. Ribozymes not only complex with target sequences via complementary antisense sequences, but also hydrolyze the target site.

Length dependence of RNA-RNA annealing.

The association of complementary nucleic acids can be described by a second order rate constant k. For extended molecules, including complex nucleic acids, values of k were shown to be proportional to the square root of the chain length L of the shorter nucleic acid strand at temperatures between tm and tm-30 degrees C. For homopolymers this is true over a wider temperature range. Below temperatures of tm-30 degrees C, annealing rate constants may sharply decrease due to the formation of intramolecular structures. It seems to be reasonable to assume that the formation of intramolecular structures of nucleic acids reduces the density of nucleation sites for annealing and, thereby, lowers the rates of association. Here, we examined the relationship between RNA chain length and the kinetics of RNA-RNA annealing at physiological ionic strength and temperature. We used a complete sequence space derived from chloramphenicol acetyltransferase (cat) sequences to average over all structures for each given length. For groups of progressively longer antisense RNA species and a 800 nucleotides long complementary RNA, the observed annealing rate constants kobs were measured in vitro. The structure-averaged values for kobs of RNA-RNA annealing were not related to the square root of the chain length. Instead, they were found to be proportional to 10(alphaL) (alpha=0.0017). Here, a theoretical model is suggested in which the observed length dependence is mainly influenced by ionic interactions between complementary RNA strands. The observed length dependence has substantial implications for the biological behavior of long-chain complementary RNA including the design of antisense RNA. The efficacy of antisense RNA in living cells is known to be related to annealing kinetics in vitro. Thus, on a statistical basis and independent of individual structures, long-chain rather than short-chain antisense RNA should lead to stronger inhibition.

Complementary large loops determine the rate of RNA duplex formation in vitro in the case of an effective antisense RNA directed against the human immunodeficiency virus type 1.

Antisense RNAs can specifically and efficiently inhibit replication of the human immunodeficiency virus type 1 (HIV-1). One of the most effective viral target regions covers the first coding exons of the viral regulator genes tat and rev. Large parts of the corresponding antisense RNAs of several hundred nucleotides in length could be removed without loss of inhibitory efficiency. The smallest antisense RNA tested (alpha Y69, 69 nucleotides in length) showed an enhanced inhibitory effect in human cells. Its secondary structure was analysed experimentally and was found to fold into a Y-shaped structure composed of two linked stem/loop structures with loop sizes of 5 and 10 nucleotides, respectively. A similar structural element was found to be formed by its complementary HIV-1-derived 645 nt long target RNA (SR6). Kinetic analyses of double strand formation between alpha Y69 and SR6 led to a second order rate constant of k = 2.9 x 10(4) M-1s-1 at 37 degrees C and physiological ionic strength. The large loop of alpha Y69 plays a crucial role in the hybridization process in vitro as shown by kinetic analyses of a set of mutants derived from alpha Y69. Base exchanges in loop regions resulted in an up to 10-fold lower association rate constant while exchanges in stem regions of alpha Y69 had no effect in vitro. We discuss a model for the pairing mechanism in vitro in which not only the first and reversible interactions (termed "kissing" in well documented cases) but also subsequent steps leading to complete RNA duplex formation make use of the large loops located at complementary positions on both RNA strands.

Kinetic selectivity of complementary nucleic acids: bcr-abl-directed antisense RNA and ribozymes.

Efficacy and sequence specificity are two major requirements in the use of antisense nucleic acids and ribozymes. For long-chain complementary RNA sequences (>30 nt), effects in living cells are correlated with the association rate of the complementary RNA in vitro, but not with the stability of the formed double strand. Thus, sequence selectivity of complementary RNA has to be defined as fast versus slow annealing with the appropriate target or non-target sequences, respectively. In this work, we performed a systematic kinetic analysis to evaluate the selectivity of bcr-abl-directed antisense RNA and hammerhead ribozymes with a length of the complementary sequences of between 20 and 80 bases. By kinetic in vitro selection, we identified oligomeric as well as long-chain complementary RNA that annealed at least tenfold faster with the bcr-abl sequence in comparison with either of the wild-type sequences bcr or abl, respectively. In the presence of selected oligodeoxynucleotide sequences and RNase H, the bcr-abl transcript was specifically hydrolysed out of a mixture containing abl and bcr sequences as well. Hammerhead ribozymes were designed such that binding with their target was facilitated either via helix I or helix III-forming antisense arms but not both. Further, cleavage and binding occurred on opposite sides of the bcr-abl fusion point. Target selectivity was found for a ribozyme that annealed fast via abl sequences and cleaved within the bcr portion of bcr-abl RNA. Kinetic probing and calculations of the local folding potential indicate that the bcr-abl fusion point sequences are not easily accessible for complementary nucleic acids. This study supports the need for more detailed structural investigations of the bcr-abl fusion sequence and forms a more rational basis for the therapeutic use of nucleic acid inhibitors of the aberrant bcr-abl gene expression in Philadelphia chromosome-positive cells.

Mechanistic insights into p53-promoted RNA-RNA annealing.

The tumour suppressor protein p53 promotes the annealing of complementary nucleic acids in vitro. We observed an up to 1600-fold increase of RNA-RNA annealing by recombinant p53 protein which was shown to bind to RNA in sequence-independent way. Nuclease mapping experiments suggest that p53 binds to intramolecular duplex portions and only marginally changes the overall secondary structure of RNA at conditions of increased annealing. Thus, the mechanism of p53-promoted RNA-RNA annealing does not seem to be dependent on an activity that melts or changes RNA structure. The activation enthalpy of RNA-RNA annealing is decreased in the presence of p53, i.e. the p53 protein could stabilize the transition state whereas the activation entropy is unfavourable. A comparison with thermodynamic data measured for other facilitators strongly suggests that the mechanism of p53-promoted RNA-RNA annealing is distinct from the mechanism by which other facilitators work. The annealing activity of p53 is almost abolished in the presence of magnesium indicating that it can be sharply regulated in vitro and, in principle, could also be regulated in vivo.

Expression of native rabbit light meromyosin in Escherichia coli. Observation of a powerful internal translation initiation site.

The cDNA-sequence coding for rabbit skeletal muscle light meromyosin (LMM) was placed under the control of the lambda promoter (PL) of an Escherichia coli expression vector. The resulting plasmid pEXLMM74 expressed non-fused rabbit skeletal muscle LMM with yields ranging from 1 to 5% of the total proteins of E. coli. This LMM was specifically recognized by polyclonal antibodies raised against chicken pectoralis muscle myosin. It could be highly enriched from E. coli extracts by using two cycles of high and low ionic strength buffer. The partially purified protein contained a major side-product, with a calculated molecular mass of 59 kilodaltons, that is produced by translation initiation from a site in the coding region of LMM. After deletion of the translation initiation site derived from the expression plasmid, only the 59 kilodalton protein is expressed in E. coli from the resulting plasmid pEXLMM59. Both the 74 and 59 kilodalton proteins were shown to form paracrystals. They were studied by electron microscopy using negative staining and were found to show characteristic striations with an axial periodicity of about 43 nm. By circular dichroism measurement we showed that the purified 59 kilodalton protein is folded mostly as an alpha-helix.

[Ki-67 antisense therapy in murine renal cell carcinoma models]. / Ki-67 Antisense-Therapie in murinen Nierenzellkarzinom-Modellen.

PURPOSE: The Ki-67 antigen is only expressed in proliferating cells. Previously, it was shown that Ki-67 derived antisense oligonucleotides (asONs) specifically inhibit the proliferation of tumor cells and tumour growth in vitro and in subcutaneous bladder and prostate tumor models. We intended to evaluate the effects of this therapeutic concept in two renal cell carcinoma (RCC) models. MATERIAL AND METHODS: Human RCC cells (SK-RC 35) were initially transfected with FITC-labeled ONs and diffferent cationic lipids to analyze the transfection efficacy by flow cytometry (FACS). The potency of 14 different ONs sequences was compared by quantitative RT-PCR in vitro. For in vivo testing, ONs were administered to immunocompetent Balb/c mice bearing orthotopic RENCA tumors as well as to SCID mice bearing subcutaneous RCC SK-RC 35 xenografts. Tumor sizes and final tumor weights were documented. Additionally, several immunohistochemical staining procedures were performed. RESULTS: FACS analysis showed highly effective transfection conditions in vitro. Systemic administration of asONs significantly decreased the tumour growth in the RENCA model (p < 0.05) and in the SCID mouse model (p = 0.009). Immunohistochemical staining of tumor specimens revealed a marked down-regulation of target protein and a slight increase in apoptotic cells after antisense treatment while the microvessel count was not significantly altered. CONCLUSION: These results demonstrate that the Ki-67 antigen represents a suitable antiproliferative target and that asONs directed against this target are potent drugs that induce a significant inhibition of renal tumor growth in different mouse models.

Theoretical and experimental approaches to design effective antisense oligonucleotides.

Among the large number of possible antisense oligonucleotides (asODN) against a given target nucleic acid, only a small number of species seems to give rise to satisfactorily strong inhibition of target gene expression in living cells. Therefore much attention is paid to strategies that help to successfully design effective asODN. Here, selected experimental approaches and theoretical concepts will be briefly described that have been developed to increase the probability of success in the use of asODN. Advantages and disadvantages of these strategies will be compared and the relatively new and controversially discussed concept of a theoretical and computer-supported design of effective asODN will be addressed.

Disinfection of cell-associated and extracellular HIV-1 by PUVA treatment.

To inactivate cell-associated and extracellular HIV-1 while preserving cellular surface antigens, a procedure was used based on PUVA treatment, i.e. addition of psoralen to cell suspensions followed by irradiation with UVA light. T-lymphoid MT-4 cells were infected with HIV-1 strain NL4-3, 4'-aminomethyl-4,5',8-trimethylpsoralen was added, and the cell suspension was irradiated with 20 mW/cm2 UVA light for 3, 4 and 5 min. To evaluate virus inactivation, cells and supernatants were diluted serially and cocultured with uninfected MT-4 cells. Infectious HIV-1 was detected by cytopathic effects, immunofluorescence and p24 antigen ELISA. UVA irradiation at 3.6 J/cm2 (3 min 20 mW/cm2) reduced the amounts of both cell-associated and extracellular infectious HIV-1 by more than five orders of magnitude. Even at more stringent conditions of PUVA treatment (10 min 20 mW/cm2 UVA irradiation), conformational cellular surface epitopes remained detectable by flow cytometry.

In vitro and in vivo action of antisense RNA.

The transient or permanent expression of antisense RNA represents one option to apply antisense techniques in biotechnology and medical research. Despite the increasing importance and use of antisense nucleic acids as well as their significant antisense-specific phenotypic effects in vivo, there is an obvious lack of explanation for the mechanism of their action. By studying naturally occurring antisense RNA and analyzing their mechanism of action we attempt to learn more about the design, the use, and the critical parameters of artificial antisense RNA. Attempts to derive models from biochemical and structural studies for the interactions between antisense RNAs and their targets will be discussed.