Dual-color simultaneous labeling of large cellular RNA

We established the high specificity and efficiency of FH14 ribozyme in labeling highly structured long RNA such as E. coli 5S rRNA. Thus, we attempted to test labeling potential of the FH14 and FJ1 ribozymes for labeling even larger cellular RNAs such as 16S and 23S rRNA (Montpetit et al., 1998; Petrov et al., 2013) (Figure 3-42). Ultimately, we sought to address both ribosomal RNAs, simultaneously, using different fluorescent dyes and both ribozymes.

These two ribosomal RNAs were chosen due to their size, structural complexity, and abundance in E. coli cells. In fact, these RNAs are so abundant that they can be visualized as two distinct bands using standard staining methods, during agarose gel electrophoresis of

total cellular RNA (Smith et al., 1988). Two potential target sites in 16S rRNA (A325 and A412), and one in 23S rRNA (A637) were targeted using FH14 alone. Three sites within 5'-RAGCY-3' context were chosen to be addressed by FJ1. Positions A272 and A1572 also situated in 5'-RAGCY-3' context, where targeted by both ribozymes (Figure 3-42).

ATTO550-ATP or 5FAM-ATP were used as labeling substrates for FH14 ribozyme and Cy5-TenDP for FJ1 ribozyme.

These two rRNAs were targeted by FH14 and FJ1 at chosen positions individually or simultaneously within the context of total cellular RNA. Due to the large size of these RNAs, agarose gel electrophoresis followed by multichannel fluorescent imaging was used for the analysis of the labeled products. All targeted positions were successfully labeled by each individual ribozyme using corresponding dye-conjugated substrate analog. In one of the samples, we tried simultaneous targeting of A649 from 16S rRNA and A272 from 23S rRNA, using FJ1 and FH14 ribozymes, respectively. As a result, 16S rRNA was labeled with Cy5 and the 23S rRNA was labeled by ATTO550, as revealed by agarose gel electrophoresis and multichannel fluorescent imaging (Figure 3-43 A).

The size difference between the two ribosomal rRNAs was enough to confirm the specificity of each ribozyme for its corresponding target sequence and cognate NTP analog.

Nevertheless, an additional confirmatory test was performed. We chose to target A1572 of 23S rRNA with FJ1 for Cy5 labeling and A412 of the 16S rRNA with FH14 for 5FAM labeling. Five reactions were prepared all containing total cellular RNA. Two of the reactions contained FJ1 ribozyme alone, with either Cy5-TenDP or 5FAM-ATP. Two other reactions were set up similarly using only the FH14 ribozyme. The 5^th reaction contained both ribozymes and both substrates. This experiment further approved the strong specificity of the ribozymes for their corresponding target sequence and their orthogonality relative to one another (Figure 3-43 B). Either 23S or 16S rRNA were labeled by their corresponding ribozymes when the correct ribozyme: dye-conjugated substrate combination was present.

In the 5^th reaction where both ribozymes and substrates were present, both ribosomal RNA were labeled with the dye assigned to their corresponding ribozyme.

Figure 3-42 Targeted positions on E. coli 16S and 23 rRNAs. Positions shaded in blue were targeted to be labeled using FJ1. Positions shaded in pink were targeted for labeling using FH14. Positions shaded in purple were targeted by both ribozymes individually. Positions marked in red, correspond to the modification site. Positions marked in green were paired to ribozyme arms. 16S and 23S rRNA secondary structures were generated by “rnacentral.org” using PDB IDs 3J9Z for16S rRNA and 4V56 for 23S rRNA.

Figure 3-43 FJ1 and FH14 mediated labeling of 16S and 23S rRNA at targeted positions. (A) Lanes 2-4: FJ1 mediated labeling of 16S rRNA at three different positions using N⁶-CyTenDP. Lanes 5-8: FH14 mediated labeling of 23S rRNA at different positions using N⁶-ATTO550-ATP. Lane 8:

corresponds to the simultaneous labeling of 16S and 23S rRNA using FJ1 and FH14 respectively.

N⁶-Cy5-TenDP was used as the labeling substrate for FJ1, whereas N⁶-ATTO550-ATP was used for FH14. Lanes 9-12: FJ1 mediated labeling of 23SrRNA at three different positions using N⁶ -Cy5-TenDP. Lanes 12 and 13: FH14-catalyzed labeling of 16S rRNA at 2 different positions using N⁶ -FAM-ATP as substrate. Lane 1 included unmodified RNA as control. The top three images were acquired using single- and multichannel fluorescent imaging of the agarose gel while the bottom gel image was acquired after SYBR Gold staining. (B) Orthogonality test of FJ1 and FH14. Four first lanes from the left include cellular RNA with either FJ1 or FH14 with cognate or orthogonal substrates. The last lane includes both ribozymes and both substrates. Successful labeling was only observed when matching ribozyme and substrate were present in the reaction. N⁶-Cy5-TenDP and N⁶-FAM-ATP were used as labeling substrates. The top three images were taken using multichannel fluorescent imaging while the bottom image was acquired after SYBR gold staining of the same gel.

Primer extension test using Superscript III reverse transcriptase and appropriate 5'-³² P-labeled primers were performed for some of the targeted sites in 16S or 23S rRNA, as was done in the case of 5S rRNA modified by FH14 and FJC9 ribozymes. For 16S and 23S rRNA however, total cellular RNA was used for modification reactions and primer extension assays. These tests confirmed the site-specificity of the FJ1 and FH14 labeling reaction for the targeted adenine nucleotides (Figure 3-44).

Figure 3-44 Reverse transcriptase primer extension assay on FJ1 or FH14 modified, 16S, and 23S rRNA. The 16S and 23S rRNA subjected to FJ1 and FH14-mediated modification in the context of total cellular RNA. N⁶-Biotin-TenDP and N⁶-biotin-ATP were used as modification substrates for FJ1 and FH14, respectively. 5'-³²P labeled primers, binding up to 40 nucleotides downstream of the modification site were used for the extension assay. A and G sequencing ladders were also included to determine the exact position of the modification site. The abortive bands appeared in every case at exactly one nucleotide 5'-to the modified position confirming site-specificity of FJ1 and FH14 ribozymes. These bands were missing in the lanes pertaining to the none-modified RNA samples.