Informational Contribution
Guide: Working with Self-Cleaving Ribozymes
Ribozymes are invaluable parts for the design of synthetic genetic circuits. They can be used in a variety of contexts, such as CRISPR/Cas-mediated applications and gene silencing. Self-cleaving ribozymes are especially handy, but can be tricky to select and use. Thus, we decided to contribute to the iGEM community by providing a guide about the theory and use regarding self-cleaving ribozymes.
1. Types and Examples
Self-cleaving ribozymes can be characterized by the position of cleavage: They either cleave close to the 5’-end of the downstream RNA sequence (5’ self-cleaving ribozyme, 5’ SCRz) [1] or close to the 3’-end of the upstream RNA sequence (3’ self-cleaving ribozyme, 3’ SCRz)[2]. The following table summarizes important information for some SCRz:
| Name | DNA Sequence | Remarks | Reference |
|---|---|---|---|
| Hammerhead Ribozyme (HHRz) | NNNNNNGACTACTCAGGCACTCCTG CTTTGCTCATTCGAGCAG | 5’ SCRz The underlined sequences are dependent on the bases that are downstream of the ribozyme. | 1, 3 |
| Type-P5 twister ribozyme | GAACGAGAGACGCAAATAGCCCGAA CTCTGGCTGCACATTTATAAATGTG CCGGCGTAATGTTC | 3’ SCRz Leaves a 5 nt long residue on the upstream RNA after cleavage. | 4 |
| Twister sister | CCTGGGCGTTCCGGCTGCCGTAGGC GGCGGCGACCACGTTCAGGTCGGCG GGGCCCCGCCCGCGAGTACCCATTTG | 5’ SCRz Leaves a 10 nt long residue on the downstream RNA after cleavage. | 5 |
| Hatchet | TTAGCAAGAATGACTATAGTCACTG TTTGTACACCCCGAATAGATTAGAA GCCTAATCATAATCACGTCTGCAAT TTTGGTACA | 3’ SCRz Leaves an 8 nt long residue on the upstream RNA after cleavage. | 1, 3 |
| Hepatitis delta virus ribozyme | CCGGCCGTACCAGGGTCGGAGGAGC GACCGCGGCCGACCCGTTGTACGAA GCCGTACCGCTTACCCTG | 3’ SCRz | 3 |
| CPEB3 | CCCCCGGTGTCGTCTTCGCAAGTGC AGCGTCGGGGACAGTCTAAGACCAC TTAGACGCTTAAGACGA | 3’ SCRz | 7 |
| Lariat capping ribozyme | GGUUGGGUUGGGAAGUAUCAUGGCU AAUCACCAUGAUGCAAUCGGGUUGA ACACUUAAUUGGGUUAAAACGGUGG GGGACGAUCCCGUAACAUCCGUCCU AACGGCGACAGACUGCACGGCCCUG CCUCUUAGGUGUGUUCAAUGAACAG UCGUUCCGAAAGGAAGCAUCCGGUA UCCCAAGACAAUC | Creates a 5’ cap-like structure at the downstream RNA. | 8 |
Please note, that these are only a few examples of self-cleaving ribozymes that we think can be very useful. There are also other self-cleaving ribozymes, e.g. the glmS ribozyme (self-cleavage + metabolite-responsive genetic switch).
2. How to get them?
We realized that it can be tricky to purify self-cleaving ribozymes from a gel after amplifying them from a plasmid via PCR. Also, it can be difficult to order them within composite DNA sequences, because they increase the complexity of the sequences. Thus, we came up with a simple solution by using a different synthesizing strategy - primer annealing. Here is a quick protocol on how to do it:
First, you need to order two oligonucleotides that are roughly the same length and overlap with 18-24 bases. To save time and money, consider to include a restriction site at the end of each oligonucleotide that you might need later. For example, you could order the oligonucleotides with BsmbI restriction sites for an ensuing golden gate assembly.
For annealing, 2 µL of the first primer/oligonucleotide (100 µM) was mixed with 2 µL (100 µM) of the primer/oligonucleotide and 6 µL water were added. This mix was incubated as shown in the table below.
| Temperature [°C] | Time |
|---|---|
3. Using them!
Creativity might be the only restriction to use self-cleaving ribozymes for genetic circuit engineering. Following, you will find examples showing some applications of ribozymes.
Abbreviations:
HHRz = hammerhead ribozyme
HDVRz = hepatitis delta virus ribozyme
A standard application is to use flanking self-cleaving ribozymes to generate precisely cut out sequences of functional RNA molecules (i.e. shRNA, saiRNA, sgRNA). This is especially useful for in vivo expression of gene editing systems (i.e. CRISPR/Cas sgRNA[3]) and inhibitory systems (i.e. saiRNA, see graphic below). The design secures the expression of functional RNA without unnecessary nucleotide residues from i.e. a terminating sequence.
Another design is for the in vivo transcription of siRNA. This design uses multiple self-cleaving ribozymes, which are all transcribed from the same gene. After cleavage, both parts of the precursor siRNA (siRNA 1 and 2) can form a double strand and act as intended [9]. Note that the spacer is necessary for the ribozymes to assume their proper secondary structure.
Further, if you want to combine self-cleaving ribozymes with protein sequences, it is important to know that the self-cleaving mechanism of the ribozymes will modify the mRNA. A 5’ SCRz will modify the downstream 5’ end of the pre-mRNA, disabling the cell from creating a 5’ cap. On the other side, a 3’ SCRz would prevent polyadenylation of the upstream pre-mRNA. Both interfere with cytoplasmic stability and translation [10]. To solve this problem, we suggest the use of a Lariat capping ribozyme (see the table further above) to replace the 5’ cap, and a synthetic poly-adenine tail to replace the missing natural poly-adenine tail.
Measurement Contribution
To assess whether glucose is affecting the formation of amilGFP, two parameters were investigated. Firstly, a growth experiment with differing glucose levels (0, 0.5, 1, 2 and 4%) was performed in a technical quadruplicate with measurements of the OD600 every 10 minutes over the curse of 20 hours shaking at 216 rpm at 37°C with two different E.coli strains (E. coli DHα, E. coli BL21 DE3).
| BL21 DE3 | max. OD600 | Time [h] | Doubling Time [h-1] | OD600 at 20 h |
|---|---|---|---|---|
| 0% | - | - | - | - |
| 0.5% | 0.369 | 7.3 | 0.05 | 0.18 |
| 1% | 0.388 | 7.1 | 0.054 | 0.24 |
| 2% | 0.389 | 6.8 | 0.057 | 0.23 |
| 4% | 0.355 | 7.3 | 0.048 | 0.2 |
BL21 showed no noteworthy difference in the doubling time with rising glucose levels and no growth without glucose.
| DH5α | max. OD600 | Time [h] | Doubling Time [h-1] | OD600 at 20 h |
|---|---|---|---|---|
| 0% | 0.043 | 10.5 | 0,003 | 0.035 |
| 0.5% | 0.369 | 8 | 0.026 | 0.054 |
| 1% | - | - | - | - |
| 2% | 0.231 | 7.3 | 0.031 | 0.11 |
| 4% | 0.249 | 7.3 | 0.031 | 0.1 |
DH5α showed no noteworthy difference in the doubling time with rising glucose levels and no growth without glucose.
Next, optical measurements for the formation of amilGFP were performed at the same conditions as mentioned above. The excitation wavelength was 485 nm and the emission wavelength was 535 nm.
10 µM Fluorescin was used as a reference and a calibration curve was prepared with a decreasing concentration (1:2 ratio per well).
| DH5α | Fluorescence at Max. OD600 |
Flourescence/time (Exponential phase) [h-1] |
Fluorescence after 20 h |
Fluorescence/time (Stationary phase) [h-1] |
|---|---|---|---|---|
| 0% | - | - | - | - |
| 0.5% | 21.25 | 2.945 | 15.5 | -0.452 |
| 1% | 22.25 | 3.133 | 24.25 | 0.155 |
| 2% | 21.75 | 3,198 | 25 | 0.246 |
| 4% | 20 | 2.739 | 20.75 | 0.059 |
The data of BL21 show that the best concentrations for the rise of fluorescence per hour are 1 and 2 % Glucose concentration. After the exponential growth phase, the rise decreases remarkably.
| DH5α | Fluorescence at Max. OD600 |
Flourescence/time (Exponential phase) [h-1] |
Fluorescence after 20 h |
Fluorescence/time (Stationary phase) [h-1] |
|---|---|---|---|---|
| 0% | 8.5 | 0.809 | 11 | 0.263 |
| 0.5% | 25.75 | 3.218 | 24 | -0.145 |
| 1% | - | - | - | - |
| 2% | 29.5 | 4.041 | 35 | 0.433 |
| 4% | 29.5 | 4.041 | 34.75 | 0.413 |
The data of DH5α show that the best concentrations for the rise of fluorescence per hour are 2 and 4% Glucose concentration. After the exponential growth phase, the rise decreases remarkably.
Although BL21 had a higher OD600 DH5α showed higher fluorescence gain per hour.
References
[1] http://labs.biology.ucsd.edu/zhao/CRISPR_web/5_prime_ribozyme_design.html
[2] http://labs.biology.ucsd.edu/zhao/CRISPR_web/3_prime_ribozyme_design.html
[3] Gao Y, Zhao Y (2014). Self-processing of ribozyme-flanked RNAs into guide RNAs in vitro and in vivo for CRISPR-mediated genome editing. Journal of Integrative Plant Biology, 56(4), 343–349. doi:10.1111/jipb.12152.
[4] Roth A, Weinberg Z, Chen AG, Kim PB, Ames TD, Breaker RR. A widespread self-cleaving ribozyme class is revealed by bioinformatics. Nat Chem Biol. 2014;10(1):56-60. doi:10.1038/nchembio.1386.
[5] Weinberg Z, Kim PB, Chen TH, et al. New classes of self-cleaving ribozymes revealed by comparative genomics analysis. Nat Chem Biol. 2015;11(8):606-610. doi:10.1038/nchembio.1846.
[6] Li S, Lünse CE, Harris KA, Breaker RR. Biochemical analysis of hatchet self-cleaving ribozymes. RNA. 2015;21(11):1845-1851. doi:10.1261/rna.052522.115.
[7] Chadalavada DM, Gratton EA, Bevilacqua PC. The human HDV-like CPEB3 ribozyme is intrinsically fast-reacting. Biochemistry. 2010;49(25):5321-5330. doi:10.1021/bi100434c.
[8] Meyer M, Nielsen H, Oliéric V, et al. Speciation of a group I intron into a lariat capping ribozyme. Proc Natl Acad Sci U S A. 2014;111(21):7659-7664. doi:10.1073/pnas.1322248111.
[9] Kato Y, Taira K. Expression of siRNA from a single transcript that includes multiple ribozymes in mammalian cells. Oligonucleotides. 2003;13(5):335-43. doi: 10.1089/154545703322617014. PMID: 15000824.
[10] Meaux S, Van Hoof A. Yeast transcripts cleaved by an internal ribozyme provide new insight into the role of the cap and poly(A) tail in translation and mRNA decay. RNA. 2006;12(7):1323-1337. doi:10.1261/rna.46306.
