What makes an rna antisense itive

The sense strand has the information that would be readable on the RNA, and that's called the coding side. The antisense is the non-coding strand, but ironically, when you're making RNA, the proteins that are involved in making RNA read the antisense strand in order to create a sense strand for the mRNA.

There's a second aspect of antisense, which is a fairly new discovery, called antisense RNA. These are RNAs that read in the opposite direction of the coding strand, and they actually bind to the coding strand of mRNAs and either target them for destruction or prevent them from being expressed.

The FLY-A13 producer cell line utilising a MLV amphotropic envelope expressor and MoMLV gag—pol expressor, 16 was used to produce replication-incompetent retroviral particles for transducing Jurkat cells in the first instance.

When cell lines stably expressing these constructs were generated in FLY-A13 cells, except for two cell lines, vector titres of only 10 3 to 10 4 c. Attempts to transduce Jurkats with all of the vectors, except pBS3sc and pBabePuro, were unsuccessful for reasons which are unclear, however, the original plasmid constructs were all successfully transfected into Jurkat cells to provide control cells for subsequent viral challenge experiments.

Proviruses derived from the vectors based on pBabePuro containing antisense cassettes in different orientations. Jurkat cells either transduced or transfected with the pBabePuro constructs were challenged with IIIB at identical challenge doses as previously Figure 5. Stable transfection with the construct pBS3P also showed inhibition of replication on viral challenge. There was no significant resistance seen in cells expressing either the L1 or L3 sequences from pBabePuro constructs data not shown.

Thus the S3 sequence consistently inhibited viral replication when expressed in T lymphocytes in a variety of different vectors including transduced or transfected murine retroviral vectors. Reverse transcriptase RT activity was measured in cell cultures until 21 days after challenge as described in Materials and methods.

This was for two reasons. First, suppression of Gag—Pol production in cells co-transfected with antisense constructs would provide supportive evidence of an inhibitory effect of these RNA sequences, in addition to viral challenge assays.

Second, analysis of cytoplasmic and virion RNA from cells transfected with these constructs might provide insights into the site of action of antisense RNA sequences in the transcription—translation pathways and subsequent steps such as RNA packaging in the case of some of the antisense sequences. Figure 6 illustrates the results of the co-transfection assays, showing that, at a ratio antisense:vector DNA , two of the five antisense constructs, L1 and L3 , and to a lesser extent S3 inhibited Gag—Pol production Figure 6a.

When these constructs were co-transfected at variable ratios to the vector construct Figure 6b , both L1 and L3 showed a dose—response relationship between the degree of inhibition and amount of antisense-expressing plasmid transfected. There was a similar, albeit less dramatic trend for S3. These results imply a significant inhibitory effect of the S3 , L1 and L3 antisense RNA sequences on the expression of Gag—Pol protein from transfected vector DNA, presumably directly or indirectly due to their antisense action.

Assuming the level of RNA transcribed is proportional to the amount of plasmid transfected, the results of co-transfection assays suggest a dose—response for L1 and L3 and not S3 and imply that the inhibitory effect of this latter molecule is maximal at a relatively low level of RNA expression. Figures represent the amalgamation of at least three separate experiments. Analysis of cytoplasmic and virion RNA from a representative co-transfection experiment by ribonuclease protection assay RPA is illustrated in Figure 7 and Table 2.

Although there is a disparity between levels of genomic RNA signal between different control samples despite equal quantities of cytoplasmic RNA being probed and then loaded it is still possible to make assessments of the effects of antisense RNA expression on the relative levels of cytoplasmic and virion vector RNA.

There was also a notable reduction in the intensity of spliced RNA bands for antisense constructs compared with sense, although relative to the amount of genomic RNA, the reductions in spliced RNA do not appear to be significant.

Therefore, while it is possible to infer that expression of each of these antisense molecules led to reduced levels of cytoplasmic genomic RNA, it is much less clear whether these RNAs had any specific effect on splicing.

Since the antisense RNAs decrease viral particle production more than can be explained by falls in encapsidation and in some cases affect cellular levels of viral mRNA, a significant action of these antisense RNA molecules seems to occur at the level of mRNA processing leading to reduced levels of both genomic and spliced RNA being exported to or surviving in the cytoplasmic compartment.

Lanes 15 and 16 represent yeast RNA controls with RNase digestion and without digestion, respectively. The primary aim of this study was to identify antisense RNA sequences able to suppress viral replication in T lymphocytes as potential antiviral genes for HIV gene therapy.

Its longer counterpart, L3 , showed moderate activity in the initial challenge assays, however, this effect was not repeated when expressed from within a retroviral vector.

These studies have raised several very important questions about antisense therapy. First, it is important to perform different studies in different model systems in order to optimise the effect. Assessment by co-transfection alone would have given misleading results and might have made it less likely that we would have considered antisense targeting the leader region downstream of the splice donor which, in effect, proved to be a consistent inhibitor of viral replication.

Second, these experiments have been done at a level of viral challenge which is considerably higher than those used in other studies. We wished to give our therapeutic molecules the most stringent test available and it is clear that the S3 antisense is capable of providing inhibition even at this very high viral challenge.

Given that the particle to infectivity ratio of HIV is something under 10 4 , the S3 antisense is clearly conferring a protective efficacy against an extremely and probably unphysiologically high concentration of infectious particles. Third, we were frustrated and surprised by the low level of expression of all of the antisenses in the cells.

They were virtually undetectable by RNase protection which means that they are being expressed at a significantly lower level than common housekeeping cellular messenger RNAs and HIV RNA which we can readily detect in infected cells. Despite this, the comparability of the RT-PCR suggests that the efficacy of the antisense is not purely a function of the level of expression.

This extraordinarily low expression together with high efficacy is paradoxically very promising information for future antisense clinical studies. Antisense RNA has emerged as an important type of inhibitor of HIV gene expression, 14 18 19 and one in vivo study, where macaques whose T lymphocytes had been transduced with antisense-expressing vectors and subsequently challenged with SIV, showed considerable promise.

The observation that each of the antisense sequences which had suppressed Gag—Pol production in the co-transfection assays reduced levels of both spliced and unspliced cytoplasmic RNA suggests a significant effect on viral RNA before translation, and is consistent with the postulated actions of antisense RNA in disrupting nuclear processing of target RNA and leading to degradation of target sequences by cellular enzymes. If, however, the results suggesting that L1 and L3 specifically inhibit genomic RNA encapsidation are significant, one might conclude that blocking this particular stage of the life cycle requires a longer antisense sequence, possibly more capable of maintaining a stable RNA duplex than shorter sequences.

As already demonstrated with antiretroviral drugs, targeting multiple stages of the HIV-1 life cycle leads to both reduced viral loads and clinical remission. Antisense RNA molecules targeting several important stages, as yet unapproachable by conventional drug therapy, expressed from a single retroviral vector may be able to induce long-term resistance to viral replication and aid in immune reconstitution. The size and positions of these sequences along with the PCR primers used to amplify them are shown in Table 1.

Recombinants containing these sequences both in the sense and antisense orientations were identified by restriction digestion and sequencing; these constructs were named pcS1A antisense orientation and pcS1S sense orientation etc.

Antisense-expressing vector constructs based on pBabePuro24 15 were constructed as follows and are illustrated in Figure 4. Cassettes were removed from the pcDNA3. Jurkat and FLY-A13 cells were selected with puromycin at 0. The titre of retroviral vector-containing supernatant on NIH-3T3 cells was measured by serial dilution. For each sample the reaction was performed in duplicate with one reaction not containing the RT enzyme negative control. Non-transfected cells were also tested as controls not shown.

The primer used for reverse transcription was complementary to the SP6 sequence downstream of the multi-cloning site in pcDNA3. Viral particles were normalised by RT activity and cellular message was normalised for total cellular RNA as previously described. Jurkat cells were challenged with HIV-1 IIIB virus stocks in a well format in order to permit large numbers of individual challenges to be performed concurrently at different concentrations of input virus.

Medium was replaced from cultures twice weekly, and from 7 days after challenge RT levels were calculated from each well twice weekly for three weeks. Swapping the base pairs at the upper stem hardly impaired the binding efficiency of the antisense RNA or its ability to inhibit repB translation in vivo or in vitro Figures 2 — 4 and Table 2.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. We thank J. Sanz for fruitful discussions and help. Ballester, S.

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