Application of the ribozymes

6.3.6.1 FH14 catalyzed labeling of the in vitro transcribed 5S rRNA and RyhB RNA

Reactions were set up with 100 pmol of either of the FH14 variants and 10 pmol of their 3'-fluorescein-labeled of their cognate transcripts (5S rRNA or RyhB). The reactions were performed in 5 µL total volume of the selection buffer including 40 mM MgCl2 and 200 µM N⁶-ATTO550-ATP. The reactions were incubated at 37°C. 1 µL aliquots were taken at 0, 2, and 4 hours and quenched by adding to 4 µL of stop solution. 2.5 µL of each of these samples was resolved on a 10% denaturing PAGE under constant power of 25 W for 1hour. The gel was then subjected to dual-channel fluorescent imaging.

For simultaneous labeling of target positions in 5S rRNA, reactions were set up with 5 pmol of the non-labeled 5S rRNA in vitro transcript and 50 pmol of FH14_5S-A45 alone, FH14_5S-A45 and FH14_5S-A66 or all three ribozymes together. The reactions were performed in 5 µL of the 1x selection buffer including 200 µM N⁶-ATTO550-ATP and 40 mM MgCl2. The reactions were quenched by adding 20 µL of stop solution after 4h incubation at 37°C. 2.5 µL of each of the quenched samples were then resolved on a 0.4 mm,

10% denaturing PAGE next to the 3'-fluorescein-labeled in vitro transcribed 5S rRNA as size marker.

6.3.6.2 Labeling 5S rRNA and RyhB RNA in total cellular RNA context

50 ng of cellular RNA was therefore mixed with 50 pmol of either of the three FH14 ribozymes in a final volume of 5 µL of the 1x selection buffer including MgCl2 and 200 µM N⁶-ATTO550-ATP. The reactions were incubated at 37°C for 4 hours before quenching by addition of 95 µL of TEN buffer followed by ethanol precipitation. The pellet was then dissolved in 2 µL of milliQ water and 2 µL of stop solution. The whole sample from all three experiments were resolved on 10% analytical PAGE for 1 hour. 1 pmol of the in vitro transcribed 3'-fluorescein-labeled 5S rRNA was also resolved on a neighboring well as a size marker. The gel was subjected to dual-channel fluorescent imaging to reveal the bands.

6.3.6.3 Mutually orthogonal double labeling of the synthetic model substrate 6.3.6.3.1 Labeling reaction

20 pmol of the double labeling substrate sequence was mixed with 50 pmol of each ribozyme in a total volume of 5 µL of selection buffer containing 40 mM MgCl2 and 200 µM of either Cy5-TenDP or 6-FAM-ATP or both. The FJ1 ribozyme was targeted towards the parent substrate sequence while the FH14 was designed to target the mutated segment. Time-point samples (0.5 µL) were taken 0, 5 h, and after overnight incubation and quenched by adding to 99.5 µL of TEN buffer. The samples were then subjected to ethanol precipitation, and the pellet was dissolved in 5 µL of the stop solution. 2.5 µL of these samples were then resolved on a 15% analytical denaturing PAGE for 45 minutes and subjected to dual-channel fluorescent imaging.

6.3.6.3.2 8-17NG mediated cleavage reaction

To perform the 8-17NG mediated cleavage experiment, an aliquot (3.5 µL) of the labeling reactions explained above was subjected to ethanol precipitation. The pellet was then dissolved in 10 µL of 1x 8-17NG reaction buffer including 10 mM MnCl2, 10 mM MgCl2,

and 200 pmol of the corresponding 8-17NG deoxyribozyme. The reaction was then incubated at 37°C and 1.4 µL timepoint samples were taken at 0, 0.5, and 1 hour and mixed

with 3.57 µL of the stop solution. 2.5 µL of these samples were resolved on the same gel as the double labeling reaction. And subjected to dual-channel fluorescent imaging.

6.3.6.4 FH14 and FJ1 mediated labeling of 16S and 23S rRNA

20 ng of total cellular RNA from Top10 E. Coli cells (NEB) was mixed with 50 pmol of the corresponding ribozyme(s). The reaction was performed in 5 µL of the selection buffer including 40 mM MgCl2 and 200 µM of the fluorescently labeled TenDP and/or ATP analogs. After 6h incubation at 37°C, the reaction was precipitated twice using isopropanol.

The resulting pellet was washed using ice cold 70% ethanol. The dried pellet was dissolved in 2 µL of stop solution and 1 µL of milliQ water. The sample was then resolved on 1.3%

agarose gel under 90 V for 45 minutes. The gel was then subjected to dual-channel fluorescent imaging. SYBR Gold staining was then performed to reveal the rest of the total cellular RNA.

6.3.6.5 Superscript III primer extension experiment

6.3.6.5.1 Preparation of the 5S rRNA modified using FH14 and FJC9 ribozymes 20 pmol of the 5S rRNA transcript was mixed with 30 pmol of either of the three 5S rRNA targeting FH14 ribozymes or the 5S rRNA targeting FJC9 ribozyme in 5 μL of 1x selection buffer including 40 mM MgCl2. In the samples in which FH14 ribozymes were present N⁶ -biotin-ATP was included at 200 μM and in the reaction in which FJC9 ribozyme was present N⁶-biotin-TenDP was used at 300 μM. The samples were incubated overnight at 37°C.

Afterwards the reactions were ethanol precipitated and the dried pellet was dissolved in 5 μL of milliQ water.

6.3.6.5.2 Preparation of the 16S and 23S rRNA modified using FJ1 and FH14

200 ng of E. coli total cellular RNA and 20 pmol of individual ribozymes were dissolved in a total volume of 10 μL of the selection buffer including 40 mM MgCl2 and the ribozyme’s cognate substrate. For FH14 type ribozyme N⁶-biotin-ATP (200 μM final concentration) was used, and for the FJ1 type ribozyme Biotin-Tenofovir-DP (340 μM final concentration). The reactions were incubated at 37°C for 5 h and were subjected to ethanol precipitation. The pellet was dried and directly used in the primer extension reaction.

6.3.6.5.3 Primer extension protocol

For primer extension experiments on the in vitro transcribed 5S rRNA, 5 pmol of the ³² P-labeled primer and 5 pmol of the modified or non-modified transcript were annealed in 5 μL of the were dissolved in 5 μL of the annealing buffer (Tris 5 mM pH= 7.5, EDTA 0.1 mM).

The sample was then placed at 95°C for 3 minutes followed by incubation at room temperature for 10 minutes. For the experiments performed on 16S and 23S rRNA, 5 pmol of the ³²P-labeled primer and 200 ng of the ribozyme-modified or non-modified total cellular RNA were annealed in 1x annealing buffer.

2 μL of the 5x First-strand synthesis buffer (Invitrogen), 0.5 μL of 0.1 M DTT, 0.5 μL of 10 mM dNTP mix and 50 units of superscript III reverse transcriptase was then added to the reactions and the final reaction volumes of were then adjusted to 10 μL by adding milliQ water. The reactions were then incubated at 55°C for 1 hour. 1 μL of 2 N NaOH was then added to each reaction and the samples were then incubated for 5 minutes at 95°C. The reactions were then ethanol precipitated. The dried pellets were dissolved in 10 μL of high dye loading buffer. 2.5 μL of each sample was resolved using 15%, analytical 45 cm long PAGE, under constant power of 45 W for 2 hours and 15 minutes. The gel was dried at 80°C under vacuum for 30 minutes and exposed to the phosphorus screen overnight. Scanning of the exposed screen was performed using Typhoon phosphorimager.

6.3.6.5.4 Sequencing ladder preparation

The sequencing reactions were performed according to (Shahn et al., 1989) with some modification. Reactions were set up by annealing 5 pmol of the ³²P-labeled primers with 5 pmol of the non-modified 5S rRNA transcript or with 200 ng of the non-modified total cellular RNA in 5 μL of the annealing buffer. for the A sequencing ladder ddTTP, was added to the reaction to a final concentration of 0.5 mM and dTTP at 0.05 mM. The rest of the dNTPs were adjusted to a final concentration of 0.5 mM. For G sequencing ladder ddCTP was included in the reaction at a concentration of 0.5 mM and dCTP at 0.05 mM. The rest of the dNTPs had a final concentration of 0.5 mM. All the other reagents such as the buffer conditions and enzyme units were the same as described for the primer extension protocol.