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. 2013 Nov 15;42(4):2473–2482. doi: 10.1093/nar/gkt1162

Figure 1.

Figure 1.

Base pairing-directed RNA primer ligation to RLuc-CVB3-CLΔ1−6 + 8 genomic RNA. (A) Schematic representation of the wt and the CLΔ1 − 6 + 8-modified CL structure. The 8-nt insertion in the 3′strand of stem A is indicated in gray. Note that T7 RNA polymerase-transcribed RNA contains two additional guanine nucleotides (italic), which form a wobble base pair with uracil nucleotides of the 3′strand of stem A in the Δ1 − 6 + 8-modified CL structure. The 12-nt RNA primer used for base pairing-directed RNA ligation is depicted in light gray, and ‘R’ represents the different 5′ modifications. (B) Urea–PAGE analysis of an RNA primer ligation to a 250-nt RNA fragment possessing the Δ1−6 + 8-mutated CL structure. RNA primer was ligated using either RNA Ligase 1 or RNA Ligase 2. Clearly, the RNA Ligase 2 was more efficient in ligating the RNA primer to the modified CLΔ1−6 + 8 structure. (C) RNA primer ligation efficiency to genomic RNA possessing the CLΔ1−6 + 8 structure was determined by urea–PAGE analysis of a 250-nt RNase H-digested 5′-terminal fragment. Note that ligation of the RNA primer reduces migration speed of the 250-nt RNase H-digested RNA fragment. (D) Translation of the incoming genomic RLuc-CVB3 RNA (wt), RNA holding the mutated CL (Δ1−6 + 8) and RNA ligation products with different 5′ ;modifications (OH, amine, biotin, Cy5) were determined by transfection of RNA in HeLa cells in the presence of GuHCl. Eight hours post-transfection, HeLa cells were lysed and RLuc values were determined. Data from a representative experiment are presented as the mean of duplicate ±SD and analyzed using unpaired t-test (** indicates significant difference P < 0.01). (E) Stability of RLuc-CVB3 RNA (wt), the mutated CL (Δ1−6 + 8) and RNA ligation product possessing a 5′hydroxyl group (OH) was followed over time. RNA was transfected into HeLa cells. At 1, 4 and 8 h post-transfection, cells were lysed and intracellular viral RNA level was analyzed using RT-qPCR. Relative RNA levels are shown in percentages compared with RLuc-CVB3 RNA (wt) at T = 1 h. (F) RNA replication of the transfected RNA was determined by comparing the RLuc values in the presence of GuHCl inhibitor (+GuHCl) and in the absence of the inhibitor (-GuHCl). Note that the extended stem of the CLΔ1-6 + 8 structure hampered RNA replication. Data from a representative experiment are presented as the mean of duplicate ±SD.