Sequence-specific Cleavage Of RNA Research Articles

CRISPR-based engineering of RNA viruses.

CRISPR RNA-guided endonucleases have enabled precise editing of DNA. However, options for editing RNA remain limited. Here, we combine sequence-specific RNA cleavage by CRISPR ribonucleases with programmable RNA repair to make precise deletions and insertions in RNA. This work establishes a recombinant RNA technology with immediate applications for the facile engineering of RNA viruses.

Selective RNA Labeling by RNA-Compatible Type II Restriction Endonuclease and RNA-Extending DNA Polymerase.

RNAs not only offer valuable information regarding our bodies but also regulate cellular functions, allowing for their specific manipulations to be extensively explored for many different biological and clinical applications. In particular, rather than temporary hybridization, permanent labeling is often required to introduce functional tags to target RNAs; however, direct RNA labeling has been revealed to be challenging, as native RNAs possess unmodifiable chemical moieties or indefinable dummy sequences at the ends of their strands. In this work, we demonstrate the combinatorial use of RNA-compatible restriction endonucleases (REs) and RNA-extending polymerases for sequence-specific RNA cleavage and subsequent RNA functionalization. Upon the introduction of complementary DNAs to target RNAs, Type II REs, such as AvrII and AvaII, could precisely cut the recognition site in the RNA-DNA heteroduplexes with exceptionally high efficiency. Subsequently, the 3′ ends of the cleaved RNAs were selectively and effectively modified when Therminator DNA polymerase template-dependently extended the RNA primers with a variety of modified nucleotides. Based on this two-step RNA labeling, only the target RNA could be chemically labeled with the desired moieties, such as bioconjugation tags or fluorophores, even in a mixture of various RNAs, demonstrating the potential for efficient and direct RNA modifications.

Selection of M2+ -Independent RNA-Cleaving DNAzymes with Side-Chains Mimicking Arginine and Lysine.

Sequence-specific cleavage of RNA by nucleic acid catalysts in the absence of a divalent metal cation (M2+ ) has remained an important goal in biomimicry with potential therapeutic applications. Given the lack of functional group diversity in canonical nucleotides, modified nucleotides with amino acid-like side chains were used to enhance self-cleavage rates at a single embedded ribonucleoside site. Previous works relied on three functional groups: an amine, a guanidine and an imidazole ensconced on three different nucleosides. However, to date, few studies have systematically addressed the necessity of all three modifications, as the value of any single modified nucleoside is contextualized at the outset of selection. Herein, we report on the use of only two modified dNTPs, excluding an imidazole, i. e. 5-(3-guanidinoallyl)-2'-dUTP (dUga TP) and 5-aminoallyl-2'-dCTP (dCaa TP), to select in-vitro self-cleaving DNAzymes that cleave in the absence of M2+ in a pH-independent fashion. Cleavage shows biphasic kinetics with rate constants that are significantly higher than in unmodified DNAzymes and compare favorably to certain DNAzymes involving an imidazole.

New Type III CRISPR variant and programmable RNA targeting tool: Oh, thank heaven for Cas7-11

Özcan etal. (2021) and van Beljouw etal. (2021) characterize a novel Type III-E CRISPR-Cas subtype, composed of a single polypeptide with crRNA processing and sequence-specific RNA cleavage activities, that provides a new RNA knockdown tool for mammalian cells with fewer off-target effects than current technologies.

Site-Selective Artificial Ribonucleases: Renaissance of Oligonucleotide Conjugates for Irreversible Cleavage of RNA Sequences.

RNA-targeting therapeutics require highly efficient sequence-specific devices capable of RNA irreversible degradation in vivo. The most developed methods of sequence-specific RNA cleavage, such as siRNA or antisense oligonucleotides (ASO), are currently based on recruitment of either intracellular multi-protein complexes or enzymes, leaving alternative approaches (e.g., ribozymes and DNAzymes) far behind. Recently, site-selective artificial ribonucleases combining the oligonucleotide recognition motifs (or their structural analogues) and catalytically active groups in a single molecular scaffold have been proven to be a great competitor to siRNA and ASO. Using the most efficient catalytic groups, utilising both metal ion-dependent (Cu(II)-2,9-dimethylphenanthroline) and metal ion-free (Tris(2-aminobenzimidazole)) on the one hand and PNA as an RNA recognising oligonucleotide on the other, allowed site-selective artificial RNases to be created with half-lives of 0.5–1 h. Artificial RNases based on the catalytic peptide [(ArgLeu)2Gly]2 were able to take progress a step further by demonstrating an ability to cleave miRNA-21 in tumour cells and provide a significant reduction of tumour growth in mice.

Characterization and Optimization of a Deoxyribozyme with a Short Left Binding Arm.

Deoxyribozymes capable of catalyzing sequence-specific RNA cleavage have broad applications in biotechnology. In vitro selected RNA-cleaving deoxyribozymes normally contain two substrate-binding arms and a central catalytic core region. Here, we describe the systematic characterization and optimization of an RNA-cleaving deoxyribozyme with an unusually short left binding arm, and its special sequence requirement for its optimal catalytic activity.

A Novel Small RNA-Cleaving Deoxyribozyme with a Short Binding Arm

Deoxyribozymes capable of catalyzing sequence-specific RNA cleavage have found broad applications in biotechnology, DNA computing and environmental sensing. Among these, deoxyribozyme 8–17 is the most common small DNA motif capable of catalyzing RNA cleavage. However, the extent to which other DNA molecules with similar catalytic motifs exist remains elusive. Here we report a novel RNA-cleaving deoxyribozyme called 10–12opt that functions with an equally small catalytic motif and an unusually short binding arm. This deoxyribozyme contains a 14-nucleotide catalytic core that preferentially catalyzes RNA cleavage at UN dinucleotide junctions (kobs = 0.9 h−1 for UU cleavage). Surprisingly, the left binding arm contains only three nucleotides and forms two canonical base pairs with the RNA substrate. Mutational analysis reveals that a riboguanosine residue 3-nucleotide downstream of cleavage site must not form canonical base pairing for the optimal catalysis, and this nucleobase likely participates in catalysis with its carbonyl O6 atom. Furthermore, we demonstrate that deoxyribozyme 10–12opt can be utilized to cleave certain microRNA sequences which are not preferentially cleaved by 8–17. Together, these results suggest that this novel RNA-cleaving deoxyribozyme forms a distinct catalytic structure than 8–17 and that sequence space may contain additional examples of DNA molecules that can cleave RNA at site-specific locations.

Structure Studies of the CRISPR-Csm Complex Reveal Mechanism of Co-transcriptional Interference

Csm, a type III-A CRISPR-Cas interference complex, is a CRISPR RNA (crRNA)-guided RNase that also possesses target RNA-dependent DNase and cyclic oligoadenylate (cOA) synthetase activities. However, the structural features allowing target RNA-binding-dependent activation of DNA cleavage and cOA generation remain unknown. Here, we report the structure of Csm in complex with crRNA together with structures of cognate or non-cognate target RNA bound Csm complexes. We show that depending on complementarity with the 5' tag of crRNA, the 3' anti-tag region of target RNA binds at two distinct sites of the Csm complex. Importantly, the interaction between the non-complementary anti-tag region of cognate target RNA and Csm1 induces a conformational change at the Csm1 subunit that allosterically activates DNA cleavage and cOA generation. Together, our structural studies provide crucial insights into the mechanistic processes required for crRNA-meditated sequence-specific RNA cleavage, RNA target-dependent non-specific DNA cleavage, and cOA generation.

Inability of DNAzymes to cleave RNA in vivo is due to limited Mg[Formula: see text] concentration in cells.

Sequence specific cleavage of RNA can be achieved by hammerhead ribozymes as well as DNAzymes. They comprise a catalytic core sequence flanked by regions that form double strands with complementary RNA. While different types of ribozymes have been discovered in natural organisms, DNAzymes derive from in vitro selection. Both have been used for therapeutic down-regulation of harmful proteins by reducing drastically the corresponding mRNA concentration. A priori DNAzymes appear advantageous because of the higher haemolytic stability and better cost effectiveness when compared to RNA. In the present work the 10-23 DNAzyme was applied to knockdown expression of the prion protein (PrP), the sole causative agent of transmissible spongiform encephalopathies. We selected accessible target sequences on the PrP mRNA based on a sequential folding algorithm. Very high effectivity of DNAzymes was found for cleavage of RNA in vitro, but activity in neuroblastoma cells was very low. However, siRNA directed to the identical target sequences reduced expression of PrP in the same cell type. According to our analysis, three Mg[Formula: see text] bind cooperatively to the DNAzyme to exert full activity. However, free ATP binds the Mg[Formula: see text] ions more strongly and already stoichiometric amounts of Mg[Formula: see text] and ATP inhibited the activity of DNAzymes drastically. In contrast, natural ribozymes form three-dimensional structures close to the cleavage site that stabilize the active conformation at much lower Mg[Formula: see text] concentrations. For DNAzymes, however, a similar stabilization is not known and therefore DNAzymes need higher free Mg[Formula: see text] concentrations than that available inside the cell.

The tRNA-Derived Small RNAs Regulate Gene Expression through Triggering Sequence-Specific Degradation of Target Transcripts in the Oomycete Pathogen Phytophthora sojae.

Along with the well-studied microRNA (miRNA) and small interfering RNA (siRNA) is a new class of transfer RNA-derived small RNA (tsRNA), which has recently been detected in multiple organisms and is implicated in gene regulation. However, while miRNAs and siRNAs are known to repress gene expression through sequence-specific RNA cleavage or translational repression, how tsRNAs regulate gene expression remains unclear. Here we report the identification and functional characterization of tsRNAs in the oomycete pathogen Phytophthora sojae. We show that multiple tRNAs are processed into abundant tsRNAs, which accumulate in a similar developmental stage-specific manner and are negatively correlated with the expression of predicted target genes. Degradome sequencing and 5′ RLM RACE experiments indicate tsRNAs can trigger degradation of target transcripts. Transient expression assays using GUS sensor constructs confirmed the requirement of sequence complementarity in tsRNA-mediated RNA degradation in P. sojae. Our results show that the tsRNA are a class of functional endogenous sRNAs and suggest that tsRNA regulate gene expression through inducing sequence-specific degradation of target RNAs in oomycetes.

Sequence-specific endoribonucleases

Ribonucleases are nucleolytic enzymes that commonly occur in living organisms and act by cleaving RNA molecules. These enzymes are involved in basic cellular processes, including the RNA maturation that accompanies the formation of functional RNAs, as well as RNA degradation that enables removal of defective or dangerous molecules or ones that have already fulfilled their cellular functions. RNA degradation is also one of the main processes that determine the amount of transcripts in the cell and thus it makes an important element of the gene expression regulation system. Ribonucleases can catalyse reactions involving RNA molecules containing specific sequences, structures or sequences within a specific structure, they can also cut RNAs non-specifically. In this article, we discuss ribonucleases cleaving the phosphodiester bond inside RNA molecules within or close to particular sequences. We also present examples of protein engineering of ribonucleases towards the development of molecular tools for sequence-specific cleavage of RNA.

Characterization of MazF-Mediated Sequence-Specific RNA Cleavage in Pseudomonas putida Using Massive Parallel Sequencing.

Under environmental stress, microbes are known to alter their translation patterns using sequence-specific endoribonucleases that we call RNA interferases. However, there has been limited insight regarding which RNAs are specifically cleaved by these RNA interferases, hence their physiological functions remain unknown. In the current study, we developed a novel method to effectively identify cleavage specificities with massive parallel sequencing. This approach uses artificially designed RNAs composed of diverse sequences, which do not form extensive secondary structures, and it correctly identified the cleavage sequence of a well-characterized Escherichia coli RNA interferase, MazF, as ACA. In addition, we also determined that an uncharacterized MazF homologue isolated from Pseudomonas putida specifically recognizes the unique triplet, UAC. Using a real-time fluorescence resonance energy transfer assay, the UAC triplet was further proved to be essential for cleavage in P. putida MazF. These results highlight an effective method to determine cleavage specificity of RNA interferases.

RNase P-Mediated Sequence-Specific Cleavage of RNA by Engineered External Guide Sequences.

The RNA cleavage activity of RNase P can be employed to decrease the levels of specific RNAs and to study their function or even to eradicate pathogens. Two different technologies have been developed to use RNase P as a tool for RNA knockdown. In one of these, an external guide sequence, which mimics a tRNA precursor, a well-known natural RNase P substrate, is used to target an RNA molecule for cleavage by endogenous RNase P. Alternatively, a guide sequence can be attached to M1 RNA, the (catalytic) RNase P RNA subunit of Escherichia coli. The guide sequence is specific for an RNA target, which is subsequently cleaved by the bacterial M1 RNA moiety. These approaches are applicable in both bacteria and eukaryotes. In this review, we will discuss the two technologies in which RNase P is used to reduce RNA expression levels.

85 Developing a new generation of peptidyl-oligonucleotide conjugates with desired biocatalytic properties against biologically relevant RNA

Sequence-specific RNA cleavage is an attractive approach for targeting specific messenger RNA sequences encoding pathogenic proteins or micro-RNAs (e.g. miR-21) up-regulated in a number of cancer p...

Sequence-specific RNA cleavage by PNA conjugates of the metal-free artificial ribonuclease tris(2-aminobenzimidazole)

Tris(2-aminobenzimidazole) conjugates with antisense oligonucleotides are effective site-specific RNA cleavers. Their mechanism of action is independent of metal ions. Here we investigate conjugates with peptide nucleic acids (PNA). RNA degradation occurs with similar rates and substrate specificities as in experiments with DNA conjugates we performed earlier. Although aggregation phenomena are observed in some cases, proper substrate recognition is not compromised. While our previous synthesis of 2-aminobenzimidazoles required an HgO induced cyclization step, a mercury free variant is described herein.

Sequence-specific cleavage of RNA by Type II restriction enzymes

The ability of 223 Type II restriction endonucleases to hydrolyze RNA–DNA heteroduplex oligonucleotide substrates was assessed. Despite the significant topological and sequence asymmetry introduced when one strand of a DNA duplex is substituted by RNA we find that six restriction enzymes (AvaII, AvrII, BanI, HaeIII, HinfI and TaqI), exclusively of the Type IIP class that recognize palindromic or interrupted-palindromic DNA sequences, catalyze robust and specific cleavage of both RNA and DNA strands of such a substrate. Time-course analyses indicate that some endonucleases hydrolyze phosphodiester bonds in both strands simultaneously whereas others appear to catalyze sequential reactions in which either the DNA or RNA product accumulates more rapidly. Such strand-specific variation in cleavage susceptibility is both significant (up to orders of magnitude difference) and somewhat sequence dependent, notably in relation to the presence or absence of uracil residues in the RNA strand. Hybridization to DNA oligonucleotides that contain endonuclease recognition sites can be used to achieve targeted hydrolysis of extended RNA substrates produced by in vitro transcription. The ability to ‘restrict’ an RNA–DNA hybrid, albeit with a limited number of restriction endonucleases, provides a method whereby individual RNA molecules can be targeted for site-specific cleavage in vitro.

A Versatile Endoribonuclease Mimic Made of DNA: Characteristics and Applications of the 8–17 RNA‐Cleaving DNAzyme

Enzymes play a crucial role in all living organisms by accelerating the rates of a myriad of biochemical reactions that are necessary to sustain life. Although the vast majority of known enzymes are made of protein, in recent years it has become increasingly apparent that other molecular formats, like nucleic acids, can also serve in this capacity. DNAzymes (also known as deoxyribozymes) are synthetic enzymes made of short, single strands of deoxyribonucleic acid. These DNA-based enzymes offer the prospect of significant commercial utility, because they are exceptionally stable and can be produced very easily and inexpensively. The study of one particular DNAzyme, known as "8-17", has enhanced our understanding of DNAzyme-mediated catalysis. Moreover, the function of 8-17 has been regarded with special importance because it can catalyze sequence-specific cleavage of RNA, a reaction that has broad implications in biotechnology and biomedical fields. In this review, we explore the creation, characterization, and application of the 8-17 RNA-cleaving DNAzyme.

Local Rather than Global Folding Enables the Lead-dependent Activity of the 8-17 Deoxyribozyme: Evidence from Contact Photo-crosslinking

The 8-17 deoxyribozyme is an in vitro selected enzyme capable of sequence-specific cleavage of RNA. While selected to be a magnesium and zinc-utilizing enzyme, the 8-17 DNAzyme has been shown to utilize lead for its catalysis. Fluorescence-based experiments have indicated that the magnesium- and zinc-utilizing versions of the DNAzyme–substrate complex need to form a defined tertiary structure to be active, but no such global folding is required for the lead-mediated activity. Here, we have investigated this phenomenon, including the use of contact photo-crosslinking to map the tertiary fold of the lead-dependent DNAzyme. While our results recapitulate that global folding is not required for the lead activity, they reveal strikingly distinct lead-mediated modes of activity under conditions of low versus moderate solution ionic strength. Even in very low salt buffers, where no global folding of the 8-17 DNAzyme occurs, the active site of the enzyme appears to form a distinct local fold, one that cannot be modelled easily by DNA/RNA constructs that preserve key sequence and secondary structure features of the active site.

Inhibition of HIV-1 replication with designed miRNAs expressed from RNA polymerase II promoters

Small interfering RNA (siRNA) mediates sequence-specific RNA cleavage and represents a potential approach to treat the infection of human immunodeficiency virus (HIV). Expression of a single siRNA species frequently led to the emergence of HIV escape variants. Thus, multiple siRNAs targeted to different regions in the HIV-1 genome may be required. However, overexpression of different anti-HIV siRNA genes from multiple pol III promoters can induce cell toxicity, thus may not be a viable option in the setting of human gene therapy trials. In the current study, we evaluated the strategy of using pol II promoters to drive the expression of siRNAs against HIV-1. We replaced the stem sequence in the stem-loop structure of the well-characterized miR-30a with siRNA sequences and showed that designed microRNA (miRNA) could be expressed from pol II promoters. We demonstrated efficient inhibition of HIV-1 replication with such designed miRNA, but the efficacy was directly correlated with the expression level. Both the vector copy number and the promoter strength directly affected the ability of the siRNA to inhibit HIV-1 replication. We also showed that a combination of pol II and pol III promoters to express two different siRNAs increased the efficacy against HIV-1 replication without comprising cell viability.

Mechanism of RNA Cleavage Catalyzed by Sequence Specific Polyamide Nucleic Acid-Neamine Conjugate

In earlier studies, we found that a conjugate of neamine-polyamide nucleic acid targeting transactivation response element of HIV-1 RNA genome (HIV-1 TAR) displayed anti-HIV-1 activity and sequence-specific cleavage of the target RNA in vitro. Here we show that both the position of conjugation of polyamide nucleic acid (PNA) on neamine and the length of the spacer are critical parameters for conferring cleavage activity to the conjugate. The conjugation of PNA via a spacer incorporating 11 atoms to the 5-position of ring I of the neamine core conferred sequence-specific RNA cleavage activity on the conjugate, while conjugation to the 4'-position of ring II abolished this activity. Similarly, 5-neamine PNA complementary to TAR sequence of HIV-1 genome (PNA(TAR)) conjugates having either a 23-atom spacer or a bulky dansyl group between PNA and the neamine core also resulted in complete loss of cleavage activity. Based on these observations, we propose a mechanism for the observed RNA cleavage catalyzed by the conjugate involving unprotonated and protonated amino groups at the 3-position of ring I and the 6'-position of ring II of the neamine core, respectively.