Catalytic RNA, ribozyme, and its applications in synthetic biology

Journal Pre-proof Catalytic RNA, ribozyme, and its applications in synthetic biology

Soyeon V. Park, Jae-Seong Yang, Hyesung Jo, Byunghwa Kang, Seung Soo Oh, Gyoo Yeol Jung PII:

S0734-9750(19)30152-1

DOI:

https://doi.org/10.1016/j.biotechadv.2019.107452

Reference:

JBA 107452

To appear in:

Biotechnology Advances

Received date:

13 May 2019

Revised date:

16 September 2019

Accepted date:

17 September 2019

Please cite this article as: S.V. Park, J.-S. Yang, H. Jo, et al., Catalytic RNA, ribozyme, and its applications in synthetic biology, Biotechnology Advances (2019), https://doi.org/ 10.1016/j.biotechadv.2019.107452

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© 2019 Published by Elsevier.

Journal Pre-proof Catalytic RNA, ribozyme, and its applications in synthetic biology

Soyeon V. Parka,1 , Jae-Seong Yangb,1, Hyesung Joa, Byunghwa Kanga, Seung Soo Oha,c,* [email protected], Gyoo Yeol Jungb,c,** [email protected]

a

Department of Materials Science and Engineering, Pohang University of Science and

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Technology (POSTECH), 77 Cheongam-Ro, Nam-Gu, Pohang, Gyeongbuk 37673, South

b

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Korea

Department of Chemical Engineering, Pohang University of Science and Technology

School of Interdisciplinary Bioscience and Bioengineering, Pohang University of Science

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c

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(POSTECH), 77 Cheongam-Ro, Nam-Gu, Pohang, Gyeongbuk 37673, South Korea

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South Korea

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and Technology (POSTECH), 77 Cheongam-Ro, Nam-Gu, Pohang, Gyeongbuk 37673,

*

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Correspondence to: S. S. Oh, Department of Materials Science and Engineering, School of

Interdisciplinary Bioscience and Bioengineering, Pohang University of Science and Technology (POSTECH), 77 Cheongam-Ro, Nam-Gu, Pohang, Gyeongbuk 37673, South Korea **

Correspondence to: G. Y. Jung, Department of Chemical Engineering, School of

Interdisciplinary Bioscience and Bioengineering, Pohang University of Science and Technology (POSTECH), 77 Cheongam-Ro, Nam-Gu, Pohang, Gyeongbuk 37673, South Korea.

1

These authors contributed equally to this work.

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Abstract Ribozymes are functional RNA molecules that can catalyze biochemical reactions. Since the discovery of the first catalytic RNA, various functional ribozymes (e.g., self-cleaving ribozymes, splicing ribozymes, RNase P, etc.) have been uncovered, and their structures and mechanisms have been identified. Ribozymes have the advantage of possessing features of "RNA" molecules; hence, they are highly applicable for manipulating various biological systems. To fully employ ribozymes in a broad range of biological applications in synthetic

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biology, a variety of ribozymes have been developed and engineered. Here, we summarize

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the main features of ribozymes and the methods used for engineering their functions. We also describe the past and recent efforts towards exploiting ribozymes for effective and novel

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applications in synthetic biology. Based on studies on their significance in biological applications till date, ribozymes are expected to advance technologies in artificial biological

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systems.

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Keywords: Ribozyme; catalytic RNA; self-cleavage; splicing; synthetic biology; expression

Abbreviations RNA DNA

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control; detecting system; logic circuit; activity regulation; gene editing

Ribonucleic acid Deoxyribonucleic acid

mRNA

Messenger RNA

HDV

Hepatitis delta virus

VS

Varkud satellite

glmS

Glucosamine-6-phosphate synthase

sTRSV

Satellite RNA associated with tobacco ringspot virus

UTR

Untranslated region

Journal Pre-proof Glucosamine-6-phosphate

RNase P

Ribonuclease P

tRNA

Transfer RNA

pre-tRNAs

Transfer RNA precursors

IGS

Internal guide sequence

EBS

Exon binding sequences

IBS

Intron binding sequences

ATP

Adenosine triphosphate

eGFP

enhanced green fluorescent protein

SD

Shine-Dalgarno

AIDS

Acquired immune deficiency syndrome

HSV-TK/GCV

Herpes simplex virus-thymidine kinase and ganciclovir

IC50

Half-maximal inhibition

ELISA E. coli Z-NCTS

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HCV

Potato spindle tuber viroid Hepatitis C virus

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PSTVd

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GlcN6P

Enzyme-linked immunosorbent assay Escherichia coli Z-stilbene linker Clustering regularly interspaced short palindrome repeat

Cas9

CRISPR/CRISPR-associated protein 9

gRNA

guide RNA

RGR

Ribozyme-gRNA-ribozyme

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1.

Introduction

Similar to protein enzymes, specific ribonucleic acid (RNA) molecules called “ribozymes” enable catalysis of a variety of biochemical reactions (e.g., cleavage and ligation of many different bonds, such as phosphodiester bonds, peptide bonds, and other chemical bonds) (Baskerville and Bartel, 2002; Doudna and Cech, 2002; Serganov et al., 2005). In 1988, Thomas R. Cech, who was awarded the 1989 Nobel Prize in chemistry for the discovery of catalytic RNAs, suggested that the catalytic functions of ribozymes would be highly valuable

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for many purposes, including analytical and biochemical studies, and that they could be

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particularly useful in medical applications (Cech, 1988). For instance, the ability to selectively cleave target RNA sequences would be directly applicable to various biochemical

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studies and viral RNA therapies, because a specific cleavage immediately disables the functions of the RNA molecules (Rossi, 1998; Yang et al., 1997). Importantly, such

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functional RNAs already exist in nature, implying that biological systems allow the ribozyme

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to be readily incorporated into their transcripts. For example, when a deoxyribonucleic acid (DNA) gene sequence is modified, a specific ribozyme can be a part of a messenger RNA

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(mRNA) or of a non-coding RNA after transcription, and this can result in the RNA transcript possessing the catalytic function of the incorporated ribozyme. Furthermore, the ribozyme

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itself can be modified to have altered functions. Thus, recent applications have proven that ribozymes can be utilized in various biological fields over and above conventional medical purposes.

As a catalytic RNA, a ribozyme has several unique advantages and characteristics over a protein enzyme (Table 1). The field of synthetic biology takes advantage of such features to build “artificial biological systems” in bioengineering research and biotechnology industry. First, a RNA transcript is functional by itself. For this reason, a ribozyme does not require a complex translation step to produce its functional form, thus allowing it to become modular. This feature is highly appealing when designing artificial biological systems because the modular ribozyme is functional within the RNA transcript (Fujita et al., 2009; Guttman and Rinn, 2012; Qin et al., 2001). Due to this modularity, we can create a logic-circuit-like construct using minimal components, and the multiple ribozymes included in the RNA transcript can perform their programmed functions simultaneously. The biological logic

Journal Pre-proof circuits can be employed as building blocks in synthetic biology, and when such building blocks are assembled systematically, desired artificial systems can be eventually created. Second, the levels of gene expression of functional RNA transcripts are more controllable and predictable than those of proteins due to the absence of a translational step. Biological circuits and tools in synthetic biology need to be accurate and robust, which can be achieved when the production levels of specific biological molecules, such as proteins or RNAs are under control. Although the levels of protein production can be predicted by translation efficiency predictors (Salis et al., 2009; Seo et al., 2013), the prediction of gene expression

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levels would be much simpler and easier. As the precise control of ribozyme expression is achievable, ribozyme-based logic circuits have the potential to provide more accurate and

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reliable performance than enzyme-based circuits while creating artificial biosystems.

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Third, transcription consumes less energy and resources than translation. Protein translation requires more than four adenosine triphosphate (ATP) molecules per amino acid, requiring an

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additional energy consumption after transcription. A typical bioengineering process involves

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a number of protein enzymes, therefore altering the metabolic pathways at the protein level often requires over-expression of several enzymes, leading to the consumption of

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exceptionally large resources (Pasini et al., 2016). Thus, efficient strategies to control biological systems would include manipulating a transcriptional process rather than

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translational or post-translational processes, and for this purpose, ribozymes are certainly an energy- and resource-efficient tool.

Forth, manipulation of ribozyme functions is straightforward. Due to the fact that the secondary structures of ribozymes are predictable, we can activate or deactivate the functions of the ribozymes by conformational reconstruction, which can be readily achieved by sequence-level binding control. For example, we can convert a natural cis-acting ribozyme to an artificial trans-acting ribozyme by splitting the original ribozyme into a catalytic core and a substrate strand (Dolan and Muller, 2014; James and Gibson, 1998). Fifth, the activity and the stability of ribozymes can be easily tuned by simple modifications. RNA is more susceptible to hydrolysis and degradation than protein or DNA. In general, RNA (median half-life, 9 h) degrades five-times faster than protein (median half-life, 46 h) in mammalian cells (Schwanhäusser et al., 2011). Although RNA has a relatively short lifetime, the feature causes high temporal expression of RNAs, which highly benefits the precise

Journal Pre-proof regulation of ribozyme activities through such temporal expression. If necessary, the life-time of a ribozyme can be easily extended in vivo by altering the base pairs or addition/deletion of stem-loops at the 5' or 3' end of RNA transcripts (Groebe and Uhlenbeck, 1988; Kobori and Yokobayashi, 2018). For the in vitro reactions, base modifications such as O-methyl modifications or linkers such as the phosphorothioate linker can be incorporated into the ribozyme to improve its stability. In view of all of the aforementioned features, the use of ribozymes can be highly beneficial to the field of synthetic biology, to aim for the creation of engineered biological systems.

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Although the first ribozyme was discovered 30 years ago, engineered ribozymes are not yet

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widely utilized in synthetic biological applications. The main researches so far have focused on discovering naturally occurring ribozymes and elucidating their catalytic structures and

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mechanisms. Based on these efforts, several ribozymes are now available, such as the hammerhead, hepatitis delta virus (HDV), and twister ribozymes. Understanding their

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structures and catalytic mechanisms eventually will lead us to engineer ribozymes that can

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have applications in the fields of molecular biology, diagnostics, and medicine. In parallel, generation of artificial ribozymes has extended the repertoire of catalytic functions of

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ribozymes. Since the time, Robertson and Joyce reported the first artificial ribozyme in 1990 using an evolutionary approach (Robertson and Joyce, 1990), in vitro selection methods have become extensively popular for identifying artificial ribozymes with novel catalytic activities

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(Popović et al., 2015; Vlassov et al., 2004). To expand the applications of ribozymes and to investigate the role of RNAs in the prebiotic environment, the evolution of catalytic RNAs has been performed in various environments, such as molecularly crowded polyethylene glycols and condensed-phase coacervates (Drobot et al., 2018; Nakano et al., 2014; Poudyal et al., 2019; Stephenson et al., 2016). As some ribozymes were even active under such unique conditions, we can also consider the possibility of discovering a completely new function of ribozymes in diverse environments. Whereas most naturally occurring ribozymes can only perform cleavage and/or ligation of a phosphodiester bond, artificial ribozymes, like protein enzymes, have been proven to perform catalytic activities to alter a broad range of chemical bonds, rendering them as promising tools to be used in the field of synthetic biology. New catalytic motifs are still being discovered in all branches of life with the help of highthroughput bioinformatics (Chen et al., 2019; Lilley, 2005; Roth et al., 2014), and novel ribozymes can be further created by innovative in vitro selection strategies (Cho et al., 2013;

Journal Pre-proof Furukawa et al., 2014; Jijakli et al., 2016). Encouraged by these outcomes, this review focuses on the most advanced biological applications of ribozymes, their properties, and engineering strategies relevant to synthetic biology.

2. Functional properties of natural and artificial ribozymes Catalytic functions of ribozymes can be generally categorized into three groups: cleavage,

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splicing, and others. Among them, the cleavage function has been most actively utilized in synthetic biology applications for re-designing an existing biological system to possess a

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specific function. To date, a variety of cleaving ribozymes have been discovered in nature, and by analyzing whether they cleave themselves or others, the cleaving ribozymes can be

2.1 Cleaving ribozymes

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2.1.1 Self-cleaving ribozymes

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further subcategorized into two sub-groups: self-cleaving and trans-cleaving ribozymes.

Self-cleaving is the most common role of naturally occurring ribozymes. These natural

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ribozymes are remarkably efficient at cleaving their own phosphodiester bonds, and this selfcleavage often plays an essential role in living organisms (e.g., multiple copy generation of viral RNAs during rolling circle replication, metabolite-responsive inhibition of gene expression, etc.) (Been and Wickham, 1997; Daros et al., 1994; Kennell et al., 1995; Winkler et al., 2004). Diverse self-cleaving ribozymes, such as the hammerhead (Ferre-D'Amare and Scott, 2010; Ren et al., 2017), hairpin (Ferre-D'Amare and Scott, 2010; Ren et al., 2017), varkud satellite (VS) (Lacroix-Labonte et al., 2016; Lilley, 2004), HDV (Duhamel et al., 1996), glucosamine-6-phosphate synthase (glmS) (Savinov and Block, 2018; Wilkinson and Been, 2005), and twister ribozymes (Roth et al., 2014) can be found in nature, and these ribozymes have been widely exploited in the field of synthetic biology. Among these ribozymes, the hammerhead ribozyme was discovered from plant pathogenic RNAs and has been known to mediate cis-cleavage of multimeric strands produced from a rolling-circle replication (Frommer et al., 2015; Muller et al., 2016). The hairpin ribozyme that originated

Journal Pre-proof from the negative strand of the satellite RNA associated with tobacco ringspot virus (sTRSV) (Hampel and Tritz, 1989) is involved in rolling circle viral replication, and shows a ligation activity as well as a self-cleavage ability. As a part of mitochondrial RNAs, the VS ribozyme was discovered from natural isolates of Neurospora, a genus of Ascomycete fungi (Collins, 2002), and the HDV ribozyme was acquired from a hepatitis delta virus (Ren et al., 2017). Similar to the role of the hammerhead and hairpin ribozymes, the activities of these ribozymes are also crucial for proliferation of organisms. Many of the gram-positive bacteria produce the glmS ribozyme, which is located at the 5'-UTR (untranslated region) of the glmS gene and catalyzes site-specific RNA cleavage in the presence of the small metabolite,

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glucosamine-6-phosphate (GlcN6P) (Winkler et al., 2004). Because the catalytic activity of

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the glmS ribozyme provides a negative feedback for the flux of GlcN6P, suggesting that the ribozyme could influence the flux of metabolites. The twister ribozyme is also one of

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naturally occurring ribozymes but was identified with the help of bioinformatics during a search of conserved RNA structural motifs (Roth et al., 2014). Unlike the roles of other

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ribozymes, that of the twister ribozyme is still being researched.

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Most of the natural ribozymes consist of multihelical junctions, pseudoknots, and nonhelical elements (Fig.1) (Jimenez et al., 2015). While the tertiary structures of the

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hammerhead, hairpin, and VS ribozymes include multiple helical junctions, those of the HDV, twister, and glmS ribozymes display additional pseudoknot structures. These complex

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structures are formed only within their own optimal conditions, strongly related to the catalytic activities of the ribozymes. Thus, it is imperative to manipulate the ribozymes toward their future uses.

The ribozymes with only multihelical junctions can be relatively easily engineered into ligand-dependent ribozymes by altering their stem sequences while preserving key structures, such as the cleavage sites of the ribozymes. For instance, the stem III sequence within the hammerhead ribozyme can be remodeled to perform ligand-dependency, but the bulge of stem I and the loop domain of stem II cannot be substituted (Fig. 1A). It is because their looploop interactions (NAN:NUN, black) are important for the hammerhead ribozyme to carry out its cleavage activity (Dufour et al., 2009). Although a ligand-specific aptamer can be grafted on stem I for the ligand-dependency, the U sequence of the bulge in stem I has to be conserved to function as a cleavage ribozyme (Beilstein et al., 2015; Win and Smolke, 2009). For the hairpin ribozyme that consists of four helices (A, B, C, and D, Fig. 1B) and loops,

Journal Pre-proof researchers have replaced or extended the sequences of helices that are less important for the overall structure (Rupert and Ferre-D'Amare, 2001; Ryder and Strobel, 2002). The interaction between loop A and loop B induces helices to stack co-axially and significantly affects the catalytic reaction. Helix C and D provide structural stability, but they are not essential for the cleavage activity. These parts, therefore, can be modified to allow the hairpin ribozyme to respond to specific metabolites as cleavage triggers (Najafi-Shoushtari and Famulok, 2007). Similar to the hairpin ribozyme, the VS ribozyme possesses a stem-loop structure (I~VI) (Fig. 1C). The key part for its cleavage reaction is the tertiary structure, wherein there is a loop-loop interaction between loop I and loop V (Lilley, 2004). Compared to the

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hammerhead and hairpin ribozymes, the VS ribozyme has been rarely used for ligand-

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dependency due to the difficulty in inserting additional bases without changing its original conformation.

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Ribozymes comprising pseudoknots (the HDV, glmS, and twister ribozymes) are more

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complex and are more difficult to be engineered because the pseudoknot structures are essential for their catalytic activities (Eiler et al., 2014; Nishikawa and Nishikawa, 2000;

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Wilkinson and Been, 2005). In general, even if trivial changes are applied to the sequences of the pseudoknots, it is difficult for the ribozymes to maintain their own functionalities. For this

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reason, scientists prefer manipulating only the parts that are irrelevant to the pseudoknots. To illustrate, the HDV ribozyme consists of five helical positions (P1.1, P1, P2, P3, and P4) and

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single-stranded regions (J1/2, L3, and J4/2) (Fig. 1D) (Jimenez et al., 2015; Shih and Been, 2002). As the four helices (P1.1, P1, P2, and P3) form an intricate pseudoknot structure, P2 helix which has little influence on the pseudoknot is modified (Beaudoin and Perreault, 2008). Similar modification methods can be applied to other pseudoknotted ribozymes as well. The glmS ribozyme, which catalyzes the cleavage activity in response to GlcN6P, has three coaxial stacking structures comprising eight helices (Cochrane et al., 2007; Klein and Ferre-D'Amare, 2006). In order to engineer the glmS ribozyme and retain its catalytic ability, its core domain (involving P2, P2.1, P2.2, P3 and P3.1) should be preserved, whereas the peripheral domain with P1 and P4 can be readily modified (Fig. 1E) (McCown et al., 2011). It should be noted that not all parts of the peripheral domain can be altered since some of them may affect the catalytic activity of the ribozyme. The twister ribozyme is another pseudoknotted ribozyme, but it is interesting to note that several of its parts allow further manipulation. This ribozyme is typically composed of four positions (P1, P2, P3, and P4) and two pseudoknots (Pk1 and Pk2) (Fig. 1F) (Eiler et al., 2014). Three locations (P1, P3, and P5)

Journal Pre-proof are known to allow sequence modification without disruption of the catalytic abilities, which is highly advantageous when conferring ligand-dependency to the ribozyme. Unlike other pseudoknotted ribozymes, we can manipulate multiple positions of the twister ribozyme simultaneously. This unique feature of the ribozyme contributed to the invention of dual ligand-responsive ribozymes (Felletti et al., 2016).

2.1.2 Trans-cleaving ribozymes

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Unlike self-cleaving ribozymes, trans-cleaving ribozymes require additional RNA

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substrates to be cleaved. These ribozymes have attracted significant attention because of their potential for sequence-specific cleavage of cellular or viral RNAs. Bacterial ribonuclease P

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(RNase P), a naturally occurring trans-cleaving ribozyme, is a large-size RNA molecule (350–410 nucleotides) that cleaves the 5'-leader sequences of transfer RNA precursors (pre-

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tRNAs) to generate mature transfer RNAs (tRNAs) (Guerrier-Takada and Altman, 2000; Navani and Li, 2006; Silverman, 2006). Although this ribozyme is comprised of an RNA

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(M1 RNA) and a polypeptide chain (C5 protein), the RNA chain alone can catalyze the transcleavage reactions. The RNA subunit of RNase P consists of two independently folding

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domains, a specificity (S) domain and a catalytic (C) domain (Torres-Larios et al., 2006). The S domain recognizes the TΨC loop structures of pre-tRNAs as a substrate of RNase P. The C

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domain guides the RNase P to 3'-CCA ends of pre-tRNAs and catalyzes the hydrolysis of 5'leader sequences. Due to self-sufficiency of the two domains, this ribozyme can be easily engineered for targeting of specific sequences and their subsequent cleavage. Because the RNase P enzyme is found in every living cell; its injection into the cell is not necessary for selective cleavage of target RNAs; however, small RNA strands should be introduced to guide RNase P to a target site that has be cleaved (Guerrier-takada et al., 1995; Yuan et al., 1992). Except RNase P, other known trans-cleaving ribozymes are created synthetically (GuerrierTakada and Altman, 2000). Most of the artificial trans-cleaving ribozymes originate from self-cleaving ribozymes, such as the hammerhead and hairpin ribozymes, because these ribozymes are relatively small and possess simple structures. For modification, the selfcleaving ribozymes are divided into two domains, a target domain that holds a cleavage site and another domain that partially hybridizes to the target domain (Saksmerprome et al., 2004;

Journal Pre-proof Schmidt et al., 1996; Webb and Luptak, 2018). For instance, the trans-acting hammerhead ribozyme (dotted box, Fig. 1A) requires modification of stem I and III to hybridize to a target RNA strand (Roychowdhury-Saha et al., 2011; Saksmerprome et al., 2004). Similarly, we can modify helix A and B of the hairpin ribozyme to trans-cleave a target RNA sequence (dotted box, Fig. 1B) (Sargueil et al., 2003; Schmidt et al., 1996). This domain-splitting strategy has been a common method for generating trans-cleaving ribozymes because it is easier to modify existing ribozymes rather than generating completely new trans-cleaving ones

Splicing ribozymes

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(Jimenez et al., 2015; Puerta-Fernández et al., 2003).

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Splicing is a post-transcriptional process for removal of introns from an RNA transcript, irrelevant to translation. For many eukaryotic cells, the splicing process is performed by a

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spliceosome, a ribonucleoprotein enzyme (Will and Luhrmann, 2011), but some functional RNAs also can self-splice their own sequences (Salldanha et al., 1993). Among these, group I

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and group II introns are the most well-characterized self-splicing ribozymes. A large number of introns belonging to these groups have been shown to catalyze such splicing reactions in

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vitro (Serganov and Patel, 2007). They join intron/exon boundaries of a precursor mRNA by

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precise excision of an intron and a subsequent covalent linking process between the exons. The secondary structure of Group I intron (Fig. 1G) has conserved structural components (P1–P10) (green) and single-stranded loop sequences (blue) (Adams et al., 2004; PuertaFernández et al., 2003). In the first step of splicing, transesterification occurs when an exogenous guanosine attacks the phosphodiester bond of U-G pair within P1 (black), and then the internal guide sequence (IGS) (orange) in the loop of P1 binds to the complementary sequence of exon 2 (orange, in exon 2). Thereafter, the second transesterification results in removal of the intron and ligation of two exons. Group II intron is a large-sized ribozyme that includes multiple stem-loop structures (Fig. 1H). It consists of six helical stems (domain D1– D6) branched from the central domain; the helix D1 and D6 are essential for the catalytic activity of the intron (Lambowitz and Zimmerly, 2011; Puerta-Fernández et al., 2003). During splicing, the conserved adenosine of D6 (black) attacks the 5'-splice site and cleaves 3'-end of exon 1 (the left red arrowhead). The cleaved end of exon 1 then attacks the 3'-splice site (the right red arrowhead) and ligates two exons. During the entire process, recognition of

Journal Pre-proof the 5'-splice site is guided by interactions between the exon binding sequences (EBS) in domain I (EBS 1 and EBS 2, orange) and the intron binding sequences (IBS) in the exon (IBS 1 and IBS 2, orange). Interestingly, the group I and II introns can be converted into transsplicing ribozymes by re-engineering essential domains, such as IGS, EBS, and IBS (Walter and Engelke, 2002). The engineered trans-splicing ribozymes showed the ability to replace a specific target RNA with a different sequence (Tanaka et al., 2017). Conventional techniques for treating genetic diseases usually utilize viral vectors (Mingozzi and High, 2011) or naked DNAs (Taniyama et al., 2002) to replace defective genes. Such methods, however, often stimulate immune responses or have low therapeutic efficacy; consequently, researchers have

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been exploring safer and more effective methods for gene therapy. In this regard, trans-

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splicing ribozymes, which can correct defective genes, present an opportunity to develop new

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medicines for gene therapy (Lan et al., 1998; Phylactou et al., 1998; Rogers et al., 2002).

Other functions

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In addition to the aforementioned ribozymes, there still exist various ribozymes that can catalyze different reactions. A ribosome is one such example (Green and Noller, 1997).

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Although a ribosome consists of RNA and protein components, ribosomal RNA is essential for translation of proteins. It can catalyze the formation of peptide bonds by joining amino

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acids, and as a result, a polypeptide chain can be synthesized. In fact, naturally occurring ribozymes found so far can catalyze only a few reactions; therefore, researchers have developed artificial ribozymes to act as new catalytic agents. For synthesizing a ribozyme having a desired catalytic function, researchers have introduced an innovative technology, in vitro selection, which identifies the desired catalytic nucleic acids from a randomized sequence pool (Tuerk and Gold, 1990; Wilson and Szostak, 1999). Since the emergence of in vitro selection techniques, researchers have invented many artificial ribozymes capable of catalyzing biochemical reactions that could not be performed by the known natural ribozymes. One of the novel functional ribozymes created by in vitro selection technology is a class I ligase ribozyme (Bartel and Szostak, 1993). Interestingly, it can catalyze the formation of phosphodiester bonds in RNA substrates. Unlike a ligase protein that needs an ATP for RNA ligation, the ligase ribozyme covalently links a substrate RNA to their own 5’ end by using a triphosphate of the ribozyme in the absence of ATP. Li and

Journal Pre-proof Huang designed a ribozyme that has a catalytic function for aminoacylation in coenzyme A (Li and Huang, 2005). The aminoacylation in coenzyme A participates in metabolic processes, including oxidative decarboxylation reactions,

oxidation of fatty acids, as well as

catabolism of amino acids. Therefore, this ribozyme can be expected to be a useful tool in controlling the relevant metabolic processes. Beyond the development of those ribozymes, researchers have consistently generated novel ribozymes that can catalyze interesting biochemical reactions, such as Diels-alder reaction (Seelig and Jaschke, 1999; Seelig et al., 2000), aldehyde reduction (Tsukiji et al., 2004), and alcohol oxidation (Tsukiji et al., 2003). Diverse catalytic functions of ribozymes allow a gene to have more functions, thus indicating

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that we could engineer synthetic biological systems possessing diverse functions in the

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future.

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3. Biological applications

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In the previous chapter, we introduced various ribozymes with different catalytic functions and interesting methods for modifying the ribozymes as desired. As mentioned earlier,

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ribozymes are RNA molecules, hence their sequences can be easily inserted into any genes, and ribozyme-including RNA transcripts can be produced. The genetic information of every

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organism is stored in genes, which is transferred to mRNAs before translation into proteins. Thus, our capability to incorporate ribozymes into diverse biological systems enables us to control cellular processes in many ways. Furthermore, the ribozymes can be applied to a wide range of biological systems, such as bacterial systems, endomembrane systems, and immunological systems, plant systems (top, Fig. 2). With all these characteristics, ribozymes have extremely broad fields of applications (e.g., clinical research, metabolic engineering, plant breeding, etc.) (Auslander and Fussenegger, 2017; Cermak et al., 2017; Jang et al., 2018; Sullenger and Gilboa, 2002). Among the many applications of ribozymes, we will further discuss the most interesting ones. The third chapter of this review will be devoted to introducing five major uses of ribozymes in synthetic biology (bottom, Fig. 2): 1) gene expression regulation, 2) detecting system, 3) logic circuit design, 4) ribozyme activity control, and 5) gene editing.

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3.1 Gene expression control A variety of ribozymes are actively used for gene expression control in the field of synthetic biology. For instance, a self-cleaving ribozyme can be engineered to respond to a specific ligand, and this ligand-dependent catalysis plays a key role in regulating essential steps in gene expression, such as initiation or termination of translation (Felletti et al., 2017; Saragliadis et al., 2013; Wieland et al., 2012). For the first time, Breaker and coworkers

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engineered a hammerhead ribozyme and generated three types of ligand-dependent ribozymes which could respond to flavin mononucleotides, theophylline, and ATP (Tang and

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Breaker, 1997a, b). By replacing the stem II of the hammerhead ribozyme (Fig 1A) with an

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appropriate ligand-specific aptamer, the Breaker group observed a modular behavior of the ribozymes, and successfully optimized the functionalities of the ligand-dependent ribozymes

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via an in vitro selection process. Surprisingly, the catalytic activities of these in vitro selected

theophylline, and ATP).

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ribozymes could be allosterically controlled by their target ligands (flavin mononucleotide,

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Other self-cleaving ribozymes can also be chosen to modulate gene expression in a ligandresponsive manner. Nomura and coworkers conducted experiments related to gene expression

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control in mammalian cells by exploiting the theophylline-responsive, self-cleaving HDV ribozyme (Nomura et al., 2013). By substituting the theophylline aptamer for the stem-loop P4-L4 of the ribozyme (Fig. 1D), the modified HDV ribozyme exhibited a catalytic function only when theophylline was bound specifically to the aptamer domain. In a model system, the engineered HDV ribozyme was inserted into the 3’-UTR of the enhanced green fluorescent protein (eGFP) gene. When theophylline was added to mammalian cells, the ribozyme within the transcribed mRNA cleaved the 3’-UTR sequence, and the theophylline-responsive cleavage impeded further translation by mRNA decay (Wang et al., 1996). This system allowed the ligand-dependent ribozyme to be utilized as a gene-OFF switch (Fig. 3A). Based on this gene-OFF switch, the ON/OFF ratio of eGFP was significantly high, up to 29.5 (Table 2). Nomura and his colleagues applied a similar strategy to create a NOR gate that could regulate translation of eGFP with two different ligands, theophylline and guanine. Thereafter, the theophylline-dependent ribozymes were further manipulated to regulate the expression of

Journal Pre-proof other genes, such as hRluc, by integrating them into the 5'-UTR of target genes (Auslander et al., 2010). Even though theophylline has been extensively used as a ligand to allosterically regulate a ribozyme activity, other small molecules are also of interest. Neomycin, for instance, was used to develop a neomycin-dependent ribozyme for gene regulation (Klauser et al., 2015). The gene-OFF switch using neomycin could control the expression of several genes (HIS3, URA3 and LacZ) when incorporated into the 3'-UTR of the Gal4 mRNA transcription factor. A number of ribozymes have been developed to respond to various small molecules, but

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typically, they do not interact with multiple ligands simultaneously. However, a ribozyme can

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be engineered to be responsive to more than two different molecules, implying that regulation of gene expression can also be simultaneously controlled by multiple ligands. For instance,

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Cho and coworkers developed a hammerhead ribozyme that simultaneously responds to hepatitis C virus (HCV) replicase and helicase, and the Breaker group designed a ribozyme

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that is dependent on both theophylline and flavin mononucleotides (Cho et al., 2005;

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Seetharaman et al., 2001). Another example is the twister ribozyme that has two modifiable substructures, thus it could be designed to hold two aptamers at different positions. This

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engineered twister ribozyme illustrated the means by which multiple ligand-controlled gene expressions using only one ribozyme could be performed (Felletti et al., 2016). Based on the

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multiple ligand-dependent ribozymes, it was possible to construct AND, NAND, OR, NOR, and NOT logic gates that use multiple ligand inputs, such as theophylline, thiamine pyrophosphate, and neomycin.

In general, gene expression systems using ligand-dependent ribozymes are usually geneOFF switches that inhibit translation through 3’-UTR or 5’-UTR cleavage of mRNA upon a target ligand (Table 2). This is because the design of the gene-OFF switch is simpler than that of the gene-ON switch when applied to a variety of genes. However, there exists an example of a gene-ON switch using a tetracycline-dependent ribozyme (Fig. 3B) (Beilstein et al., 2015). Here, the cleavage activity of the ribozyme was inhibited when tetracycline bound to the ribozyme, subsequently promoting gene expression. Unfortunately, the ON/OFF ratio of gene expression level was relatively low; the gene-ON switch exhibited an ON/OFF ratio of approximately 3.5 ~ 4.8, which was much lower than that of typical gene-OFF switches (Table 2). Consequently, the poor ON/OFF ratio and complication in design makes the geneON switch unpopular.

Journal Pre-proof In addition to small molecules, RNA molecules can act as triggers for manipulating the gene expression (Vauleon and Muller, 2003). For instance, a target RNA strand complementary to the stem III sequences of the hammerhead ribozyme (Fig. 1A) could deactivate the selfcleaving activity of the ribozyme (Klauser and Hartig, 2013). In this study, within the mRNA transcript, the stem I of the hammerhead ribozyme harbored the Shine Dalgarno (SD) region, so that the ribosomes were inaccessible to the SD region without self-cleavage. In the absence of the target RNAs, self-cleavage of the ribozyme immediately unlocked the SD region for subsequent translation process. However, this self-cleavage function was inhibited due to the hybridization of the target strand with the stem III region of the ribozyme

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sequence. Therefore, in the presence of the target RNAs, SD region remained inaccessible to

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the ribosomes, resulting in inhibition of translation.

The aforementioned methods of regulating gene expression by such engineered ribozymes

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have a potential for controlling enzyme levels and metabolic flux. If we employ metabolic

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intermediates as ligands for engineered ribozymes to manipulate a specific metabolic pathway in microorganisms, we can intentionally increase the amount of a desired compound,

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which is otherwise generated in small quantities via the natural pathway (Niu et al., 2018; Stevens and Carothers, 2015). Such metabolic engineering can be applied for microbial

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production of compounds and proteins that have a high demand in the biotechnology industry. Additionally, living organisms inhibit the generation of toxic compounds and

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control homeostasis via metabolic pathways. Imitating natural homeostatic systems and constructing a novel anti-toxic system is one of the major challenges that the field of metabolic engineering is facing. To regulate the metabolites via desired pathways, we have to control the enzyme expression systems. Ribozymes, which are based on RNA, are effective tools for such applications in regulating gene expression. Although several reports have proven that such a ligand-dependent cleavage system can be successful in regulating gene expression, it still needs to be further improved. Integration of the appropriate ribozyme sequence into a desired gene has been tedious so far. Moreover, developing a new ligand-dependent ribozyme has been very onerous and time-consuming (Win and Smolke, 2009; Wittmann and Suess, 2011). To overcome these limitations, the number of steps to produce the ligand-dependent ribozymes should be reduced by applying improved technologies. For example, various in vitro selection technologies (Cho et al., 2013; Cho et al., 2010; Oh et al., 2011; Qu et al., 2016) have dramatically improved systematic

Journal Pre-proof search for specific purposes, and such techniques would have a potential to considerably reduce the laborious processes employed for synthesizing optimal artificial ribozymes.

3.2 Detecting system Diverse factors can seriously influence metabolism in living organisms. For instance, erroneous protein expression cause malfunctions in physiological responses (Furugian et al.,

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1991; Saltiel and Kahn, 2001; Singh et al., 2004). Besides, pathogenic organisms and other external factors induce a variety of syndromes (e.g., Ebola hemorrhagic fever, Acquired

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immune deficiency syndrome (AIDS), and poisoning)(Baize et al., 2014; Burtonboy and Debruyere, 1985; Duruibe et al., 2007). Even therapeutic drugs can cause toxicity if they are

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administered at a dose beyond the therapeutic index (Muller and Milton, 2012). Therefore,

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detecting such sources of toxicity is often important to make engineered organisms viable in various environments (Pardee et al., 2014). Towards this, researchers in the field of synthetic

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biology have designed several detection systems using target-activated ribozymes. Initially, small molecule detection systems based on

ribozymes activated by target

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molecules (Hartig et al., 2002; Kennedy et al., 2014; Seetharaman et al., 2001; Silverman, 2006; Vaish et al., 2002) were utilized for the detection of target-activated ribozymes. When

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a cell itself can detect the target, it can be engineered to perform feedback reactions, such as regulation of an operon for cell suicide. For example, based on a theophylline-dependent ribozyme, an engineered mammalian cell could detect theophylline molecules, resulting in the operation of a suicide gene system, Herpes Simplex Virus-Thymidine Kinase and Ganciclovir (HSV-TK/GCV) system (Zhang et al., 2017). In the presence of theophylline, the ribozyme cleaved itself and suppressed the expression of the HSV-TK gene. As a result, production of cytotoxic molecules was also suppressed, resulting in high cell viability. In order to measure HSV-TK gene expression, the eGFP gene was incorporated between the HSV-TK gene and the sequence of theophylline-dependent ribozyme (HSV-TK gene-eGFP gene-ribozyme), and fluorescence of eGFP was measured. The level of eGFP expression with theophylline was 4.1-fold lower than that without theophylline, confirming lower expression of HSV-TK proteins. Another example of small molecule detectors is a neomycin-induced, protein expression system (Klauser et al., 2015). Neomycin is an aminoglycoside antibiotic,

Journal Pre-proof and the neomycin-dependent hammerhead ribozyme was introduced in a bacteria cell to regulate expression of a transcription factor, Gal4. Interestingly, only 0.7 µg/mL neomycin showed half-maximal inhibition (IC50) of expression of Gal4. Given that the maximum amount of neomycin used for pharmacological studies in humans is as high as 4.3 µg/mL (Samuel and Waithe, 1961), this system holds enormous potential as a sensitive neomycin detector. To detect a protein target, a combination of a protein-specific aptamer and a self-cleaving ribozyme has been suggested. In a study by Smolke and colleagues, the high-affinity MS2

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protein aptamer was replaced with the stem I or II of the hammerhead ribozyme (Fig. 1A) originating from the sTRSV (Kennedy et al., 2014). The Smolke’s group enabled the sTRSV

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hammerhead ribozyme to control its cleavage activity depending on MS2 and applied the modified ribozyme to yeasts and mammalian cells to detect the MS2 protein in vivo. This

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MS2 detection system could either act as a gene-ON or a gene-OFF switch; it was rationally

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designed to activate or inhibit expression of GFP gene upon MS2 protein levels. The fluorescent intensity of GFP increased from 20 to 97% of the maximal GFP level using the

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gene-ON switch, whereas it decreased from 55 to 13% with the gene-OFF switch.

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Similar to the small molecule and protein detection systems described above, a virus detection system has been described by Tien and colleagues, based on a target-responsive,

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cleaving ribozyme (Yang et al., 1997). To detect potato spindle tuber viroid (PSTVd), this group created a trans-acting hammerhead ribozyme that could detect and cleave a specific RNA region of PSTVd. If potatoes are genetically modified to include the hammerhead ribozyme, propagation of the incorporated PSTVd should be disabled. Indeed, accumulation of PSTVd in the potato that included this ribozyme was 20-fold less compared to that in the control, indicating that the transgenic potatoes were rendered PSTVd-resistant. Motivated by this result, Gago and colleagues developed vegetables in which PSTVd accumulation was inhibited by using modified hammerhead ribozymes (Carbonell et al., 2011). Unlike common detection systems based on cleaving-ribozymes, an interesting system uses a ligase ribozyme to detect viruses (Kossen et al., 2004). This artificial ligase is a truncated form of the class I ligase ribozyme and can be activated only when bound to specific nucleic acid strands. In the study, the target oligoribonucleotides represented the conserved sequences of the HCV genome, triggering the ligation activity of the ribozyme. Because the ligation activity was negligible without the target RNA strands, the ligase ribozyme-based

Journal Pre-proof detection method was extremely sensitive to detect even zeptomole (10-21 mole) amounts of target strands. Typically, direct monitoring of ligation activity is laborious and timeconsuming; hence, secondary signals of ligation are often measured instead. Accordingly, two different detection systems were developed as easy-to-use monitoring methods; one was an Enzyme-linked immunosorbent assay (ELISA) that exploits anti-fluorescein antibodies conjugated to alkaline phosphatase, and the other was an optical immunoassay. In the ELISA method, when the target-activated ribozyme ligates to an RNA substrate carrying fluorescein, an anti-fluorescein antibody binds to the fluorescein, and this binding induces an enzymatic reaction with alkaline phosphatase. The fluorescence intensity of the reaction product can

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thus be measured for the target strand detection. This ELISA was sensitive and produced a

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distinct signal only with 1.6 attomole target HCV strands (330 fM). In the optical immunoassay-type method, the target-activated ribozyme ligates to a biotinylated substrate

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immobilized on a surface; and subsequently, an avidin-conjugated horseradish peroxidase is used. The captured peroxidase converts 3,3’,5,5’-tetramethylbenzidine molecules into an

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insoluble product, and this precipitated product results in a color change. Although this method showed a similar detection sensitivity as that of the ELISA-type method, it has the

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advantage wherein the color change is visible to the naked eye.

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Similar to the real targets above, environmental conditions, such as temperature can also be detected by ribozyme-containing cell systems and can be used to control cell functions. For

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example, some hammerhead ribozymes were combined with the RNA thermometer to be utilized as a heat-sensing system (Rossmanith and Narberhaus, 2016; Saragliadis et al., 2013). Previously, the RNA thermometer was developed to work as a hairpin strand that can selectively block the SD region in a temperature-responsive manner (Chowdhury et al., 2003; Storz, 1999). This hairpin strand formed a duplex with the SD region at low temperature, whereas high temperature denatured the duplex, leading to initiation of translation. Narberhaus and co-workers exploited the theophylline-dependent ribozyme and RNA thermometer to control translation, depending on temperature and theophylline molecules (Rossmanith and Narberhaus, 2016). Two different gene expression switches were prepared; the first switch required both theophylline and high temperature (37°C) to initiate translation, the second one did not require the theophylline molecule but required a high temperature. At low temperature (25°C), both the switches allowed the RNA thermometer to block the SD region. These switches could act as both thermal sensors and logic gates using the thermal input. A different method to use the hammerhead ribozyme for development of thermal

Journal Pre-proof sensors has been proposed (Saragliadis et al., 2013). In this study, a ribozyme was engineered to have a heat-controlled cleavage function. Specifically, a temperature-dependent hairpin sequence was inserted in the stem III region of the hammerhead ribozyme (Fig. 1A); the heatdependent deformation of the U-rich hairpin structure was observed to strongly correlate with the catalytic activity of the ribozyme. Because the hairpin structure within stem III could not be formed at a high temperature (42°C), the ribozyme was inhibited to perform a cleavage reaction, retaining the blocked SD region. However, a low temperature (19°C) allowed the formation of the hairpin structure, which led to the catalytic cleavage and subsequent

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unlocking of the SD region that initiated the translation process.

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3.3 Logic circuit design

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As a component of logic circuit, a logic gate processes inputs and outputs using a binary system. In general, the logic gate is applied to save data and convert inputs to outputs in

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electric devices (Fredkin and Toffoli, 1982). Interestingly, researchers in the field of synthetic biology have extended the usage of logic gates to synthetic biological circuits that modulate

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cellular functions and responses (Hasty et al., 2002; Knight and Sussman, 1998; Kylilis et al., 2018; Zeng et al., 2018). Ribozymes are attractive synthetic biological components for such

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purposes; engineered ribozymes can simply extract outputs (catalytic functions) from inputs (ligands), and more importantly, incorporating the engineered ribozymes into a cellular system is relatively easy. For instance, Klauser and coworkers invented two-input gates that could control protein translation in Escherichia coli (E. coli) by utilizing two switches comprised of engineered ribozymes (Klauser et al., 2012). One of two switches controlled accessibility of ribosomes to a ribosome binding site using a thiamine pyrophosphateinduced, self-cleaving ribozyme; consequently, initiation of translation depended on thiamine pyrophosphates. The other switch possessed a theophylline-dependent, self-cleaving ribozyme for modulating the activation of specific tRNA that recognizes amber (UAG) stop codons, and termination of translation could be controlled by the theophylline ligands. A combination of these two switches produced several logic gates (AND, NOR, and NAND gates) by using both thiamine pyrophosphate and theophylline as inputs, and as an output, gene expression was regulated at the post-transcriptional level. Despite low ON/OFF ratios of

Journal Pre-proof these logic gates, they have successfully proven the feasibility of employing ribozyme-based logic circuits for synthetic biology. Moreover, an antisense RNA complementary to portions of the ribozyme can be employed as an input of logic circuit systems. For instance, Penchovsky studied digital circuits that include ribozymes that are allosterically controlled by antisense RNAs (Penchovsky, 2012). The allosteric ribozymes were engineered to have binding sites of several antisense RNAs, which can activate the catalytic function of the ribozymes. As a result, various logic circuits, such as a three-input AND logic gate and a two-input multiplexer were developed. These

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antisense RNA-based logic circuits are advantageous because one can freely alter the

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antisense RNA sequences and their binding positions within the allosteric ribozymes. Gander and his colleagues developed a NOR gate that regulates the expression of target

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gene in yeast cells by using clustering regularly interspaced short palindrome repeat (CRISPR)/dead CRISPR-related protein 9 (dCas9) proteins and self-cleaving ribozymes

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(Gander et al., 2017). When two different dCas9 proteins were added as inputs, the

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transcription of the logic circuit was suppressed due to steric hindrance of RNA polymerase by dCas9 proteins. Since the RNA polymerase no longer transcribed the mRNA that included

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cleaving ribozymes and a guide RNA (gRNA) of the target gene, final gRNA-dCas9 complexes of the target gene were not generated, leading to the expression of target gene.

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This study is an interesting example, which demonstrates that biological logic circuits can be developed in a eukaryotic system as well as a prokaryotic system. From these studies, we can anticipate that creating advanced ribozymes would actualize development of more complex devices, which could be used to control various biological systems.

3.4 Ribozyme activity regulation Several biological applications such as gene therapy could be improved if ribozyme activities are easily controlled (Li et al., 2006). The catalytic activities of ribozymes generally rely on their own tertiary structures such as loop-loop interactions, pseudoknots, and helical junctions (Ferre-D'Amare and Scott, 2010; Ren et al., 2017), and thus, researchers have tried to modify these tertiary structures to control these activities. For instance, Nakatani’s group intentionally attenuated a loop-loop interaction of the hammerhead ribozyme, and if

Journal Pre-proof necessary, the weakened interaction could be intensified by the assistance of naphthyridine carbamate tetramer with Z-stilbene linker (Z-NCTS) (Dohno et al., 2018). They reduced the loop-loop interaction of the ribozyme by replacing the CAA/UAC sequences of the loop-loop region (between stem I and stem II, Fig. 1A) with G-rich mismatch sequences (AGG/UGG). Because of this replacement, the cleavage activity of the ribozyme was effectively inhibited. Upon adding Z-NCTS to the inhibited ribozyme, the hydrogen bond between the four naphthyridine moieties and guanine bases of AGG/UGG formed loops that bound to each other. As a result, cleavage activity of the ribozyme was restored. When the loop-loop interaction was recovered, the cleavage efficiency of the ribozyme increased up to 80%

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depending on the amount of Z-NCTS. As another example of regulation of ribozyme activity,

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tris(2-aminoethyl) amine (tren)-derived scaffolds have been exploited (Mao et al., 2017). The stem II sequence of the hammerhead ribozyme (Fig.1 A) were replaced with the U- rich

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sequence, resulting in the loss of ribozyme activity by the stem unfolding. However, when a tris(2-aminoethyl) amine (tren)-derived scaffold with two (t2M) and four (t4M) melamine

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rings was added to the inactive ribozyme, the melamine rings interacted with the uracils to

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refold the stem structure, leading to recovery of its catalytic activity. In addition, some investigators have attempted to control the activity of ribozyme using light

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energy (Velema et al., 2018). The 2'-OH group of RNAs was acylated with a bulky group (dimethoxy-2-nitrobenzyl 2-chloro-1H-imidazole-1-carboxylate), which could be removed by

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irradiation with UV light (365 nm). When the 2′-OH moieties of an RNA molecule were replaced by photo-responsive groups, its ability to hybridize with the complementary strand was disabled. This light-controlled hybridization was applied to the folding of the hammerhead ribozyme. Cleavage activity of the ribozyme with the photo-responsive groups showed a higher inhibition (370 times) than that of the wild type ribozyme. However, when the ribozyme was exposed to UV light, it almost recovered its maximum activity, exhibiting similar cleavage efficiency as that of the wild type ribozyme.

3.5 Gene editing CRISPR/CRISPR-associated protein 9 (Cas9) system is an innovative tool for selective cleaving of specific genes for genome editing (Hsu et al., 2014; Ran et al., 2013). This system

Journal Pre-proof consists of two components, the Cas9 protein and the gRNA. Although many researchers have improved the CRISPR/Cas9 system to manipulate the genome of various organisms, the level of its safety, fidelity, and accuracy in editing is not yet satisfactory to be applied for manipulation of human genomes (Kosicki et al., 2018; Shin et al., 2017). Moreover, the current gRNA-producing method based on RNA polymerase III and its promoters (U3 and U6) has several critical limitations (Gao and Zhao, 2014; Lee et al., 2016). Since all eukaryotic cells express the RNA polymerase III, we cannot restrict production of gRNAs to specific cells or tissues. Furthermore, we have not fully identified the U3/U6 promoter

essential for gRNA production is not available in market.

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sequences of commercially valuable organisms, and unfortunately, the RNA polymerase III

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To overcome these limitations, Gao and Zhao have demonstrated a new strategy that utilizes ribozymes as producers of gRNAs (Gao and Zhao, 2014). They introduced a specifically

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designed gene sequence called Ribozyme-gRNA-ribozyme (RGR) gene (Fig. 4A). When

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transcribed, the gRNA sequence was inserted between two different self-cleaving ribozymes, the hammerhead and HDV ribozymes. As a result, self-cleaving events occurred at both ends

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of the RGR, releasing the gRNA strand. The unbound gRNA then directed the Cas9 protein to a target gene for editing. Because this RGR approach does not strongly depend on the use

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of RNA polymerase III and U3/U6 promoters, it is possible to use different types of polymerases, such as RNA polymerase II. The choice of flexibility allows us to employ

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specific promoters for transcription of gRNAs (e.g., tissue-specific promoters and ligandinducible promoters), making this approach feasible for many different organisms. Moreover, multiple RGR sequences can be designed to target various genes simultaneously, because transcription of RGRs is initiated concurrently with an identical promoter sequence. Otherwise, simultaneous targeting of multiple genes would be impossible. Due to these advantages, the RGR method has become more popular in the field of gene editing (Lee et al., 2016; Li et al., 2018; Marshall et al., 2017; Tang, X. et al., 2017; Yoshioka et al., 2015). On-demand control of cleavage activities could be one of key solutions to enhance safety of genome editing by reducing off-target gene cleavage. However, based on the current CRISPR/Cas9 system, the catalytic function of Cas9 cannot be easily controlled because the Cas9-gRNA complex can retain its cleavage activity even after successful cleavage of the target genes (Clarke et al., 2018). Although disassembly of gRNA and Cas9 may deactivate the reaction of the system, it has been significantly difficult to detach the gRNA from the

Journal Pre-proof complex. To address this difficulty, researchers devised different approaches to form the gRNA/Cas9 complex on demand and suggested the ribozyme-induced gRNA release as an alternative (Tang, W. et al., 2017). In one study, a theophylline-induced, self-cleaving ribozyme was designed to be incorporated into 5’ end of gRNA, which effectively blocked the spacer region (target-specific binding region) of the gRNA (top, Fig. 4B). Since the blocked gRNA could not recognize the complementary site of the target gene, the activity of Cas9 was suppressed in the absence of theophylline. However, introduction of theophylline induced a self-cleaving reaction of the incorporated ribozyme (middle, Fig. 4B), thereby unblocking the gRNA to direct Cas9 to the target gene. As a model for this system, the GFP

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genes of HEK293 cells were induced to be cleaved. Addition of theophylline successfully

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caused a loss of fluorescence of GFPs; the decrement of the GFP fluorescence was four-fold higher than that of the control group, which harbored inactive ribozymes. This result

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indicates that the strategy of gRNA release induced by ribozymes achieved on-demand control of Cas9 activity in mammalian cells. Also, a guanine-dependent ribozyme was

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explored, and it successfully regulated expression of GFP. These results imply that the strategy of ribozyme-induced gRNA activation can be a general methodology. In other words,

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utilizing other ligands might prove to be helpful to control gene editing functions. Despite the success of this technology, it still has a series of setbacks; for instance, a low level of

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cleavage activity is detected even in the absence of ligands (Beilstein et al., 2015; Nomura et al., 2013; Soukup and Breaker, 1999) and the presence of ribozymes, in some way, decreases

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the editing efficiency. Without solving these issues, safety of genome editing cannot be guaranteed; thus, more advanced and reliable editing technologies have to be developed.

4. Further challenges and perspectives In this paper, we have reviewed the characteristics of natural and engineered ribozymes, and their past and recent developments were further investigated in a variety of biological applications. To date, ribozymes have been developed for ligand-triggered gene expression regulation in particular and occasionally for medical/environmental detection. In this regard, we have reviewed interesting examples; however, there is still room for further advancement in this field.

Journal Pre-proof One of the main issues and challenges in further development of ribozyme-based biological applications is to expand the catalytic diversity of ribozymes. Although a large number of natural and artificial ribozymes have been discovered, the repertoire of catalytic functions of ribozymes is still mainly limited to cleavage or ligation of phosphodiester bonds. To fully use the ribozymes in a broad range of biological applications in synthetic biology, it would be necessary to further develop ribozymes with new catalytic functions, such as mediating metabolic reactions. Despite few examples, some researchers believe RNA-based metabolic reaction systems would be achievable (Chen et al., 2007; Ralser, 2014). Ribozymes that catalyze metabolic reactions in the absence of protein enzymes might be more efficient and

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controllable. Thus, such ribozymes could be used to produce high-value products compared

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to those produced by protein enzyme-based systems. Moreover, availability of in vitro transcription would facilitate the mass production of ribozymes to be used in industry.

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However, it would be considered in advance that the catalytic activities of ribozymes should

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be sufficiently high even in a harsh condition, such as in chemical fermentation. Another issue is that only a few ligands are available to control catalytic functions of

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ribozymes. Currently, most researches use theophylline and flavin mononucleotide, but there is a need to explore additional ligands (e.g., disease-related molecules) and the ribozymes

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specific to those ligands. If available, the new ribozymes can be promising tools for detecting fatal diseases and environmental pollutants, such as harmful microbes, toxins, and heavy

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metals. Identifying new ligands of natural ribozymes to regulate their functions will provide opportunities to discover novel antibiotics and drugs. For example, when bacteria develop antibiotic resistance against conventional drugs, the ligands that control glmS ribozymes can behave as novel antibacterial molecules (Ferré-D’Amaré, 2010). Apart from addressing these issues, the main advantages of ribozymes over protein enzymes also need to be considered. Although evolutionary selection processes have been applied to the generation of catalytic proteins and peptides (Arnold, 2018; Kuruma and Ueda, 2015; Pappas et al., 2016), their selection is not as easy as that of ribozymes because of several complex steps. As ribozymes possess both a genotype and phenotype, their evolved catalytic properties are directly correlated with their specific genetic changes, which can be easily identified by various sequencing methods (Mardis, 2008; Wu et al., 2014). Due to simple in vitro transcription and reverse transcription, the isolated ribozymes can be readily amplified during in vivo and in vitro selection processes. Unfortunately, there are no protein

Journal Pre-proof amplification techniques till date, and identification of amino acid sequences is technically challenging, making the evolution process of protein enzymes highly complicated. For example, selection methods, such as compartmentalization ribosome display and RNA display essentially require functional parts (proteins/peptides) to be linked to their own coding parts (nucleic acids) for further amplification and identification of isolated enzymes (Hanes and Plückthun, 1997; Roberts and Szostak, 1997). Moreover, the intermolecular interactions between RNAs are highly predictable. Unlike protein enzymes, ribozymes can be rationally designed to form assemblies of multiple

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ribozymes (Ohuchi et al., 2002; Rahman et al., 2017). For example, Ikawa’s group has studied a self-assembly of group I ribozymes that consist of catalytic core modules and

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activator modules (Rahman et al., 2018a, b). Although the two modules interact with each other for self-assembly, Ikawa and co-workers covalently linked the two modules. As a

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result, a number of group I ribozymes were assembled together by intermolecular interactions

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of multiple modules. Surprisingly, the assembled ribozyme complex showed a greater catalytic activity than the single ribozyme. Current development of RNA self-assembly and

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origami technologies offer a variety of unique architectures (Chworos et al., 2004; Dibrov et al., 2011; Ishikawa et al., 2013), and based on them, it is possible to build self-assembling

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modular ribozyme systems, which could further enhance catalytic activities for various

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biological applications, including regulatory systems. The recent development of computational and experimental selection strategies for ribozyme activities has led to the creation of new functions and new ligands for ribozymes (Kobori et al., 2017). Furthermore, with the emerging next-generation sequencing technologies, we can now characterize each variant of a large library from either in vitro selection pools or exceptionally large parallel experiments, and the resulting data are highly valuable to understand the strong relationships among ribozyme sequences, structures, and functions. For example, Kobori et. al comprehensively studied all three base pairs in P4 (Fig. 1D) proximal to the ribozyme core of the HDV-like ribozyme (total 4096 variants) and discovered that the ribozyme activity is highly sensitive to the sequences and the apparent stabilities (Kobori and Yokobayashi, 2018). From this study, the variants of ribozyme with different activities were successfully prepared and used to fine-tune gene expression levels in mammalian cells. Currently, RNA structure prediction has become more accurate, and new methods have been developed to infer the secondary structures of RNA molecules and to

Journal Pre-proof determine their crystal structures (Miao and Westhof, 2017; Mortimer et al., 2012; Ritchey et al., 2017; Strobel et al., 2018; Zinshteyn et al., 2019). Several algorithms are helpful to design RNA sequences, which form a desired secondary structure at equilibrium (Zadeh et al., 2011). In addition to these computational methods, researchers have developed novel experimental methods to characterize secondary structures of RNA by integrating highthroughput chemical probing, massive sequencing, and computational analysis (Ge and Zhang, 2015; Loughrey et al., 2014). In particular, the next generation sequencing technology can be highly useful to obtain massive information related to RNA structures, such as paired and the unpaired bases. As a result, even in vivo secondary and tertiary structures of RNA can

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be speculated with high accuracy. We believe that combining all these advanced technologies

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in ribozyme selection, sequencing, and structure analysis, there would be an opportunity to create a powerful tool to effectively and efficiently generate a variety of high-performance

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ribozymes, greatly expanding the applications of the ribozymes.

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In addition, we anticipate that advanced bioinformatics will play a critical role in developing diverse ribozymes with various capabilities. We are aware that many naturally occurring

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ribozymes originate from non-mammalian systems, and artificial ribozymes, such as aptazymes have been developed from in vitro processes or from prokaryotic systems. Then,

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how can we make sure that such ribozymes truly work in mammalian systems? We cannot determine whether a specific ribozyme is appropriate for a certain synthetic biology

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application without having information about properties of ribozymes (e.g., responses of ribozymes to different environmental conditions, such as pH, temperature, and metal ion concentrations and catalytic kinetics depending on different host cells). For example, it is known that mobile bacterial group II introns, such as ribozyme L1.LtrB do not function at low Mg2+ concentrations in mammalian cells, so we avoid their use in mammalian gene therapy (Truong et al., 2015). Thus, we should systematically survey and collect information about various properties of ribozymes. Furthermore, a database like BioBrick (Shetty et al., 2008) would be especially helpful. Currently, a community-wide popular database of ribozymes has not yet been developed, but there are several ongoing attempts to prepare databases that collate and assemble information about all ribozyme/aptamer sequences and their detailed annotation (e.g., RiboaptDB and Aptamer Base) (Cruz-Toledo et al., 2012; Thodima et al., 2006). These efforts will accelerate the emergence of ribozyme-based, synthetic biology applications, including development of general methods to convert the

Journal Pre-proof existing ribozymes suitable for application to mammalian systems (Kobori and Yokobayashi, 2018). Since this review focuses on the role of ribozymes and catalytic RNAs in biotechnology, we did not mention the other types of RNAs, such as aptamers and riboswitches that have been successfully utilized in a variety of biotechnological applications (Chappell et al., 2015; Liang et al., 2011). Unlike ribozymes, they are non-catalytic RNAs that cannot modulate the cleavage or formation of chemical bonds. For example, aptamers are short, single stranded nucleic acids specialized for molecular binding to various ligands with high binding affinity

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and specificity. Like aptamers, riboswitches also bind to targets, such as metabolites; however, they additionally perform binding-induced conformational changes. In nature,

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organisms have already employed riboswitch-based gene regulatory systems in mRNA transcripts. Moreover, researchers have used non-catalytic RNAs for biotechnological

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applications, such as repressing the overexpression of toxic molecules and activating

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expressions of proteins, depending on their environmental conditions, such as binding of metabolites and change in temperature (Chappell et al., 2015; Liang et al., 2011). As these

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aptamers and riboswitches are RNAs, they can be easily incorporated in a single RNA transcript along with a ribozyme for regulating ribozyme activities or vice versa (Wilkinson

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and Been, 2005).

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We have witnessed that the combination of ribozymes and other RNA-based synthetic biology tools, such as CRISPR/Cas9 system affords great opportunities for exploring novel applications (Gao and Zhao, 2014; Lee et al., 2016). When a ribozyme was combined with the CRISPR/Cas9 system, a conditional gene knock-out system was eventually created. Similarly, ribozymes can be combined with other functional systems to create RNA devices with complex functionalities. For example, RNA localization motifs were integrated with ribozymes, and the resulting RNA device could be delivered into specific sites of cells. An RNA device would provide therapeutic use when localized with virus RNAs (Hormes et al., 1997; Khan, 2006). In general, RNA devices are modular, and their functions can be predictable in terms of secondary sequence-structure relationship. However, some parts may interact with others to exhibit undesired effects. In order to actualize the combination between ribozymes and other functional systems, it would be necessary to develop computational tools that support the successful design of complex RNA devices. With the vast data obtained from all high-

Journal Pre-proof throughput experiments and profound learning of artificial intelligence, the computational design in the future will reach the extremely high levels of complexity, thus enables automatic arrangement of programmable ribozymes for desired complex functions. With a lot of effort put into discovering natural and artificial ribozymes, the expansion of novel catalytic functions is ongoing (Higgs and Lehman, 2015; Müller, 2015; Nelson and Breaker, 2017). It is worth mentioning once again that the ribozymes have their own unique advantages in synthetic biology; because of their small size and modularity in design, the ribozymes are typically independent of transcription factors and are used to control gene

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expression with different small molecules. If they are further developed to have improved activities and orthogonality in different reactions, advancement of emerging RNA-based

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biotechnology will gain more momentum. As we still do not fully understand the biological roles of all ribozymes, ceaseless efforts to study them across diverse organisms may provide

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insights to exploit and improve the ribozymes in future.

Acknowledgements

2017R1C1B3012050)

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This work was supported by the National Research Foundation of Korea (NRF) grant (NRFand

the

Global

Research

Laboratory

Program

(NRF-

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2016K1A1A2912829) funded by the Korea government (Ministry of Science and ICT). Also, this research was supported by Basic Science Research Program through the National Research

Foundation

of

Korea

(NRF)

funded

by the

Ministry of

Education

(2018R1A6A3A11045727).

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Journal Pre-proof Table 1. Comparison between ribozymes and protein enzymes Ribozyme

Protein Enzyme

Require transcription

O

O

Require translation

X

O

Modular design

Easy

Medium

Expression control

Relatively easy

Medium

Structure prediction

Medium

Hard

Sequence-based interaction control

Relatively easy

Hard

Catalytic function

Limited

Diverse

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Property

Engineered ribozyme

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Table 2. Characteristics of gene-switches based on cleaving ribozymes Ligand

Type of switch

Regulated gene

Cell types

ON/OFF ratio

Reference

Hammerhead

theophylline

OFF

egfp, hRluc

mammalian cell

16~22

(Auslander et al., 2010)

Hammerhead

theophylline

ON

egfp

E. coli

10

(Wieland et al., 2012)

Hammerhead

theophylline

OFF

SEAP

mammalian cell

4

(Wieland et al., 2012)

Hammerhead

theophylline, neomycin

OFF

HIS3, URA3, LacZ

yeast

up to 25

(Klauser et al., 2015)

Hammerhead

tetracycline

ON

egfp, hRluc

mammalian cell

3.5~4.8

(Beilstein et al., 2015)

Hammerhead

small RNA

OFF

egfp

E. coli

10

HDV

theophylline, guanine

OFF

egfp

mammalian cell

29.5

(Nomura et al., 2013)

Twister

theophylline

OFF

egfp

E. coli

6~10

(Felletti et al., 2016)

Twister

thiamine pyrophosphate

OFF

egfp

E. coli

5~35

(Felletti et al., 2016)

Twister

neomycin

OFF

LacZ

yeast

10

(Felletti et al., 2016)

(Klauser and Hartig, 2013)

Journal Pre-proof Figure Captions Fig. 1. Secondary structures of self-cleaving ribozymes (A: Hammerhead, B: Hairpin, C: VS, D: HDV, E: Twister, and F: glmS) and self-splicing ribozymes (G: Group I intron and H: Group II intron). Helical stems, loops, and pseudoknots are marked in green, blue, and yellow, respectively. Each ribozyme has its own cleavage site indicated by red arrowheads. Cleavage sites have consistent sequences (red) important for catalytic activities of ribozymes (N: any nucleotide; H: A, C or U). Among the self-cleaving ribozymes, the hammerhead (A) and hairpin ribozymes (B) have been engineered to possess trans-acting structures, as shown

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in the dotted boxes. Group I and II introns require IGS and EBS (orange) to bind to target domains within the exons, to recognize splice-sites. For splicing mechanisms, certain

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sequences should be conserved (a U-G pair, G, and A, black).

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Fig. 2. An illustration of the major applications of ribozymes in the field of synthetic biology. Engineered ribozymes can be incorporated into a variety of biological systems (e.g., bacterial

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systems, endomembrane systems, immune systems, plant systems, etc.), which has a potential for interesting synthetic biological applications, such as gene expression regulation, target

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detection, logic circuit, ribozyme activity control, and gene editing.

Fig. 3. Regulatory mechanisms of eGFP gene expression switches. (A) A theophyllinedependent ribozyme is incorporated into a mRNA transcript and triggers the cleavage activity of the ribozyme, resulting in inhibition of eGFP gene expression. (B) The mRNA transcript that includes a tetracycline-dependent ribozyme cannot express the eGFP gene in the absence of tetracycline due to self-cleavage of the incorporated ribozyme. However, when a tetracycline molecule binds to the ribozyme, the cleavage activity becomes inactive, and the eGFP gene is subsequently expressed. Ligand-binding regions are shown as orange lines.

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Fig. 4. Strategies of ribozyme-mediated gRNA generation in CRISPR/Cas9 gene editing. (A) gRNA self-generation of RGR gene. A gRNA sequence (blue) is located between the hammerhead (left green square) and HDV ribozymes (right green square). When both the ribozymes catalyze self-cleaving reactions, a gRNA is released, and a Cas9-gRNA complex is formed to edit target genes. (B) On-demand gRNA generation. The end of gRNA sequence is flanked with a target-induced, self-cleaving ribozyme, which is inactive in absence of a target. Upon introduction of the target, folding of target-binding region (orange) enables the

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ribozyme to catalyze its cleavage reaction. After the cleavage, the generated gRNA is

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assembled with Cas9, and the Cas9-gRNA complex edits target genes.

Journal Pre-proof Highlights Unique features of catalytic RNAs over protein enzymes



Characteristics of ribozymes and their engineering strategies



Interesting applications of ribozymes in the field of synthetic biology



Current challenges of ribozyme-based biotechnology and future perspectives

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Figure 1

Figure 2

Figure 3

Figure 4