mRNA fusion constructs serve in a general cell-based assay to profile oligonucleotide activity

Abstract

A cellular assay has been developed to allow measurement of the inhibitory activity of large numbers of oligonucleotides at the protein level. The assay is centred on an mRNA fusion transcript construct comprising of a full-length reporter gene with a target region of interest inserted into the 3'-untranslated region. Luciferase and fluorescent reporter genes were used in the constructs. The insert can be from multiple sources (uncharacterised ESTs, partial or full-length genes, genes from alternate species, etc.). Large numbers of oligonucleotides were screened for antisense activity against a number of such constructs bearing different reporters, in different cell lines and the inhibitory profiles obtained were compared with those observed through screening the oligonucleotides against the corresponding endogenous genes assayed at the mRNA level. A high degree of similarity in the profiles was obtained indicating that the fusion constructs are suitable surrogates for the endogenous messages for characterisation of antisense oligonucleotides (ASOs). Furthermore, the results support the hypothesis that the secondary structure of mRNAs are divided into domains, the nature of which is determined by primary nucleotide sequence. Oligonucleotides whose activity is dependent on the local structure of their target mRNAs (e.g. ASOs, short interfering RNAs) can be characterised via such fusion RNA constructs.

Figure 1

Schematic drawing of the construction of a fusion mRNA and the mechanism of action of an oligonucleotide targeting the fusion mRNA. (A) A fusion mRNA is generated from a plasmid in which an EcoRI/NotI cDNA fragment is inserted into the reporter gene. Thus, a target RNA region is created in the 3′-UTR of the mRNA encoding the reporter protein (luciferase or fluorescent protein). (B) Two examples of reporter constructs produced from endogenous mRNAs are shown. In example 1, a full-length cDNA is cloned into the vector to produce the fusion mRNA, whereas in example 2, only a part of a cDNA, e.g. EST, is cloned into the vector. Both types of constructs have been used as target sequences. (C) The ASO binds to the endogenous mRNA as well as to the fusion RNA in the same local environment, leading to message degradation due to RNase H cleavage.

Figure 1

Schematic drawing of the construction of a fusion mRNA and the mechanism of action of an oligonucleotide targeting the fusion mRNA. (A) A fusion mRNA is generated from a plasmid in which an EcoRI/NotI cDNA fragment is inserted into the reporter gene. Thus, a target RNA region is created in the 3′-UTR of the mRNA encoding the reporter protein (luciferase or fluorescent protein). (B) Two examples of reporter constructs produced from endogenous mRNAs are shown. In example 1, a full-length cDNA is cloned into the vector to produce the fusion mRNA, whereas in example 2, only a part of a cDNA, e.g. EST, is cloned into the vector. Both types of constructs have been used as target sequences. (C) The ASO binds to the endogenous mRNA as well as to the fusion RNA in the same local environment, leading to message degradation due to RNase H cleavage.

Figure 1

Schematic drawing of the construction of a fusion mRNA and the mechanism of action of an oligonucleotide targeting the fusion mRNA. (A) A fusion mRNA is generated from a plasmid in which an EcoRI/NotI cDNA fragment is inserted into the reporter gene. Thus, a target RNA region is created in the 3′-UTR of the mRNA encoding the reporter protein (luciferase or fluorescent protein). (B) Two examples of reporter constructs produced from endogenous mRNAs are shown. In example 1, a full-length cDNA is cloned into the vector to produce the fusion mRNA, whereas in example 2, only a part of a cDNA, e.g. EST, is cloned into the vector. Both types of constructs have been used as target sequences. (C) The ASO binds to the endogenous mRNA as well as to the fusion RNA in the same local environment, leading to message degradation due to RNase H cleavage.

Figure 2

Detached map of the reporter plasmids pNAS-020, pNAS-055 and pNAS-092 (see also Table 1). The cloning site adjacent to the STOP codon of the firefly luciferase (pNAS-020) and the eYFP (pNAS-055 and pNAS-092) for cDNA or EST inserts is illustrated in the separate box. Amp, ampicillin selection marker; Zeo, Zeocin selection marker; CMVmin, minimal human cytomegalovirus promoter; CMV, human cytomegalovirus promoter; EF-1a, elongation factor 1 alpha promoter; T7-prom, bacterial T7 promoter; SplD-BG, splicing donor site of rabbit β-globin; SplA-GB, splicing acceptor site of rabbit β-globin; pA-BG, polyadenylation site of rabbit β-globin; pA-SV40, polyadenylation site of simian virus 40; pA-BGH, polyadenylation site of bovine growth hormone; pBRori, origin of replication of pBR322; SV40ori, SV40 origin of replication. StuI-XhoI-fill, NheI-HindIII-fill, HindIII-BamHI-fill, NotI-StuI-fill, SmaI-NotI-fill, NotI-XhoI-fill, filled in Klenow-blunted ligation sites used for vector constructions.

Figure 2

Detached map of the reporter plasmids pNAS-020, pNAS-055 and pNAS-092 (see also Table 1). The cloning site adjacent to the STOP codon of the firefly luciferase (pNAS-020) and the eYFP (pNAS-055 and pNAS-092) for cDNA or EST inserts is illustrated in the separate box. Amp, ampicillin selection marker; Zeo, Zeocin selection marker; CMVmin, minimal human cytomegalovirus promoter; CMV, human cytomegalovirus promoter; EF-1a, elongation factor 1 alpha promoter; T7-prom, bacterial T7 promoter; SplD-BG, splicing donor site of rabbit β-globin; SplA-GB, splicing acceptor site of rabbit β-globin; pA-BG, polyadenylation site of rabbit β-globin; pA-SV40, polyadenylation site of simian virus 40; pA-BGH, polyadenylation site of bovine growth hormone; pBRori, origin of replication of pBR322; SV40ori, SV40 origin of replication. StuI-XhoI-fill, NheI-HindIII-fill, HindIII-BamHI-fill, NotI-StuI-fill, SmaI-NotI-fill, NotI-XhoI-fill, filled in Klenow-blunted ligation sites used for vector constructions.

Figure 2

Detached map of the reporter plasmids pNAS-020, pNAS-055 and pNAS-092 (see also Table 1). The cloning site adjacent to the STOP codon of the firefly luciferase (pNAS-020) and the eYFP (pNAS-055 and pNAS-092) for cDNA or EST inserts is illustrated in the separate box. Amp, ampicillin selection marker; Zeo, Zeocin selection marker; CMVmin, minimal human cytomegalovirus promoter; CMV, human cytomegalovirus promoter; EF-1a, elongation factor 1 alpha promoter; T7-prom, bacterial T7 promoter; SplD-BG, splicing donor site of rabbit β-globin; SplA-GB, splicing acceptor site of rabbit β-globin; pA-BG, polyadenylation site of rabbit β-globin; pA-SV40, polyadenylation site of simian virus 40; pA-BGH, polyadenylation site of bovine growth hormone; pBRori, origin of replication of pBR322; SV40ori, SV40 origin of replication. StuI-XhoI-fill, NheI-HindIII-fill, HindIII-BamHI-fill, NotI-StuI-fill, SmaI-NotI-fill, NotI-XhoI-fill, filled in Klenow-blunted ligation sites used for vector constructions.

Figure 3

Correlation plot comparing the effect of 58 transfected ASOs on endogenous mRNA levels in human H-1299 cells and the effect on fusion mRNA of interest expressing the reporter in SSF-3-G cells. A series of eight targets were compared derived from ESTs. Correlation coefficient r² = 0.73 (linear least-squares regression). Dashed lines indicate the 95% confidence level for the prediction. The circled area indicates the region of highly active ASOs. (Triangles) Most potent hits in the reporter assay from each target. (Grey circle) Second-best ASO in the reporter assay corresponding to the grey triangle. The white triangle outside of the circled area belongs to a target against which no better ASO was found in the q-PCR. (Crosses) Three values outside the 95% confidence level.

Figure 4

Correlation plot comparing the effect of 32 transfected ASOs on eYFP reporter constructs (construct type pNAS-055) bearing the fusion RNA of interest in human H-1299-C-TR cells versus the effect on luciferase reporter constructs (construct type pNAS-020) bearing the fusion RNA of interest in SSF-3-G cells. Two EST targets were compared. Correlation coefficient r² = 0.75 (linear least-squares regression). Dashed lines show the 95% confidence level for the prediction. The cross indicates one value outside the confidence level.

Figure 5

Out of 20 ASOs, the four most potent were identified in a fluorescent protein reporter screen using a derivative of pNAS-055. Subsequently, these four ASOs and their corresponding mismatches (mm) were re-tested at 400 nM concentration using either activity of the reporter construct in H-1299-C-TR cells (activity measured 48 h after transfection) or in H-1299 cells, by measuring the expression level of the endogenous mRNA activity assayed by q-PCR (RNA isolated 24 h after transfection). Pairwise correlation was found between both RNA targets, the model fusion mRNA and the endogenous mRNA. From a correlation plot of the various activities a correlation coefficient of r² = 0.86 was calculated (linear least-squares regression).

Figure 6

Dose–response effect of three transfected ASOs tested on reporter construct type pNAS-092. COS-1 cells were transfected and the read-outs after 72 h are shown; plasmid transfection using Fugene™ reagent.

Figure 7

Ninety six single well screening of match and mismatch ASO pairs on reporter construct type pNAS-092 with COS-1 cells after 48 h of ASO transfection (150 nM); plasmid transfection with Fugene. Match ASOs are on the left side of the figure. Four nucleotide mismatch controls to the first 30 ASOs are shown on the right side. From left to right, the first depicted mismatch sequences correspond to the first match sequences, etc. Grey bars represent the most potent oligonucleotides. The 100% of control black bar from each plate represents the average of 26 untreated plasmid wells with the indicated relative error bar of 12.7 and 6.3%, respectively. (A) Treatment of the reporter bearing the fusion RNA of interest with ASOs. (B) Treatment of a control plasmid of an unrelated RNA fusion with ASOs.