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.

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.

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.

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.

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.

Up to 100-fold increase of apparent gene expression in the presence of Epstein-Barr virus oriP sequences and EBNA1: implications of the nuclear import of plasmids.

A 100-fold increase in luciferase activity was observed in 293 cells, stably expressing Epstein-Barr nuclear antigen 1 (EBNA1; 293-EBNA1 cells), that had been transiently transfected with plasmids carrying Epstein-Barr virus (EBV) oriP sequences. This increase was observed in comparison to reporter gene activity obtained after transfection with a plasmid carrying no oriP sequences. The luciferase gene on these plasmids was under the control of either the cytomegalovirus immediate-early 1 gene enhancer-promoter (CMV IE1) or the Rous sarcoma virus promoter. The increase of reporter gene activity was not due to plasmid replication, since a similar enhancement was observed in the presence of aphidicolin, an inhibitor of replicative DNA synthesis, or after deletion of the dyad symmetry (DS) element within oriP. Luciferase production was not increased in the presence of only the DS element. Microinjection of plasmids carrying the CMV IE1 promoter-driven luciferase gene with or without oriP sequences into the nuclei of 293-EBNA1 cells resulted in a 17-fold increase in luciferase activity. Cytoplasmic injection of these plasmids led to an enhancement of luciferase activity of up to 100-fold. This difference in the factor of activation after nuclear or cytoplasmic injection could be ascribed to increased transport of plasmids carrying oriP from the cytoplasm to the nucleus in the presence of EBNA1. These data suggest the possibility of substantially increasing the apparent expression of a gene under the control of a strong constitutive promoter in the presence of oriP sequences and EBNA1. This improvement in expression is due to intranuclear enhancement of gene expression. oriP-specific transport of plasmid DNA from the cytoplasm of 293-EBNA1 cells to the nucleus seems to contribute to the observed effect.

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.

Theoretical and experimental selection parameters for HBV-directed antisense RNA are related to increased RNA-RNA annealing.

Annealing kinetics of antisense species against two different target regions of the hepatitis B virus (HBV) were measured by kinetic in vitro selection. Individual association rates were related to energies calculated for local sequence segments and predicted structures of the complete pregenomic target RNA. A relationship between the presence of external loops and joint sequences with fast pairing was observed whereas internal loops did not favor fast RNA-RNA annealing. The findings were used to predict a fast-annealing HBV-directed antisense oligodeoxyribonucleotide that turned out to pair with its target RNA at an association rate constant of k=9.2 x 10(4) M(-1) s(-1), which is substantially faster than the annealing rates of artificial antisense RNA so far included in in vitro selection assays.

The hepatitis B virus posttranscriptional regulatory element contains a highly stable RNA secondary structure.

Hepatitis B virus (HBV) transcripts contain a sequence known as the posttranscriptional regulatory element (PRE). This element was shown to facilitate the nuclear export of the S gene transcripts, to partially substitute for the human immunodeficiency virus (HIV-1) Rev-response element (RRE), and to bind two cellular factors. Within the genetically defined PRE (approximately 450 nucleotides), we identified a highly stable secondary structural element of 313 nucleotides in length termed PRE313. The energy values of the PRE313 are similar to those of the RRE of HIV-1 and significantly lower than those of other portions of HBV RNA. A comparison of human HBV subtypes shows strong conservation of the PRE313 in terms of energy and structure, providing further evidence for the biological significance of the genetically defined PRE and the PRE313 in particular. The structural model for the PRE313 described in this study may help in identifying crucial components of the transport mechanism of transcripts of HBV.

Methylation sensitivity of the enhancer from the human papillomavirus type 16.

The human papillomavirus type 16 is associated with anogenital cancer. Transcription of the viral transforming genes E6 and E7 is under the control of an epithelial cell type-specific enhancer. In the enhancer core, we have identified a regulatory element that is recognized by a novel nuclear factor named MSPF (methylation-sensitive papillomavirus transcription factor). Mutating the MSPF binding site strongly affects the enhancer activity. The MSPF recognition sequence 5'-ATGCGNNNNCGCCT-3' contains two CpG dinucleotides, potential targets for 5-cytidine methylation. DNA recognition by MSPF is strictly methylation-sensitive, since introduction of 5-methylcytidine into either CpG abolishes complex formation. Moreover, CpG methylation of the MSPF binding site suppresses the activity of the enhancer and of the MSPF enhanson subfragment in vivo. In the cervical carcinoma cell line CaSki, which has integrated multiple transcriptionally inactive human papilloma virus 16 genomes, a few of the viral genomes are methylated at the MSPF binding site. These findings suggest that viral transcription can be suppressed by methylation of the regulatory region, an event that prevents binding of the cellular transcription factor MSPF.