Sequence-specific Cleavage Activities Research Articles

Emergence of catalytic function in prebiotic information-coding polymers

Life as we know it relies on the interplay between catalytic activity and information processing carried out by biological polymers. Here we present a plausible pathway by which a pool of prebiotic information-coding oligomers could acquire an early catalytic function, namely sequence-specific cleavage activity. Starting with a system capable of non-enzymatic templated replication, we demonstrate that even non-catalyzed spontaneous cleavage would promote proliferation by generating short fragments that act as primers. Furthermore, we show that catalytic cleavage function can naturally emerge and proliferate in this system. Specifically, a cooperative catalytic network with four subpopulations of oligomers is selected by the evolution in competition with chains lacking catalytic activity. The cooperative system emerges through the functional differentiation of oligomers into catalysts and their substrates. The model is inspired by the structure of the hammerhead RNA enzyme as well as other DNA- and RNA-based enzymes with cleavage activity that readily emerge through natural or artificial selection. We identify the conditions necessary for the emergence of the cooperative catalytic network. In particular, we show that it requires the catalytic rate enhancement over the spontaneous cleavage rate to be at least 102–103, a factor consistent with the existing experiments. The evolutionary pressure leads to a further increase in catalytic efficiency. The presented mechanism provides an escape route from a relatively simple pairwise replication of oligomers toward a more complex behavior involving catalytic function. This provides a bridge between the information-first origin of life scenarios and the paradigm of autocatalytic sets and hypercycles, albeit based on cleavage rather than synthesis of reactants.

Emergence of catalytic function in prebiotic information-coding polymers.

Life as we know it relies on the interplay between catalytic activity and information processing carried out by biological polymers. Here we present a plausible pathway by which a pool of prebiotic information-coding oligomers could acquire an early catalytic function, namely sequence-specific cleavage activity. Starting with a system capable of non-enzymatic templated replication, we demonstrate that even non-catalyzed spontaneous cleavage would promote proliferation by generating short fragments that act as primers. Furthermore, we show that catalytic cleavage function can naturally emerge and proliferate in this system. Specifically, a cooperative catalytic network with four subpopulations of oligomers is selected by the evolution in competition with chains lacking catalytic activity. The cooperative system emerges through the functional differentiation of oligomers into catalysts and their substrates. The model is inspired by the structure of the hammerhead RNA enzyme as well as other DNA- and RNA-based enzymes with cleavage activity that readily emerge through natural or artificial selection. We identify the conditions necessary for the emergence of the cooperative catalytic network. In particular, we show that it requires the catalytic rate enhancement over the spontaneous cleavage rate to be at least 102-103, a factor consistent with the existing experiments. The evolutionary pressure leads to a further increase in catalytic efficiency. The presented mechanism provides an escape route from a relatively simple pairwise replication of oligomers toward a more complex behavior involving catalytic function. This provides a bridge between the information-first origin of life scenarios and the paradigm of autocatalytic sets and hypercycles, albeit based on cleavage rather than synthesis of reactants.

Prediction of the sequence-specific cleavage activity of Cas9 variants.

Several Streptococcus pyogenes Cas9 (SpCas9) variants have been developed to improve an enzyme's specificity or to alter or broaden its protospacer-adjacent motif (PAM) compatibility, but selecting the optimal variant for a given target sequence and application remains difficult. To build computational models to predict the sequence-specific activity of 13 SpCas9 variants, we first assessed their cleavage efficiency at 26,891 target sequences. We found that, of the 256 possible four-nucleotide NNNN sequences, 156 can be used as a PAM by at least one of the SpCas9 variants. For the high-fidelity variants, overall activity could be ranked as SpCas9 ≥ Sniper-Cas9 > eSpCas9(1.1) > SpCas9-HF1 > HypaCas9 ≈ xCas9 >> evoCas9, whereas their overall specificities could be ranked as evoCas9 >> HypaCas9 ≥ SpCas9-HF1 ≈ eSpCas9(1.1) > xCas9 > Sniper-Cas9 > SpCas9. Using these data, we developed 16 deep-learning-based computational models that accurately predict the activity of these variants at any target sequence.

Sequence-Specific Cleavage of BM2 Gene Transcript of Influenza B Virus by 10-23 Catalytic Motif Containing DNA Enzymes Significantly Inhibits Viral RNA Translation and Replication

One of the hallmarks of progression of influenza virus replication is the step involving the virus uncoating that occurs in the host cytoplasm. The BM2 ion channel protein of influenza B virus is highly conserved and is essentially required during the uncoating processes of virus, thus an attractive target for designing antiviral drugs. We screened several DNA enzymes (Dzs) containing the 10-23 catalytic motif against the influenza B virus BM2 RNA. Dzs directed against the predicted single-stranded bulge regions showed sequence-specific cleavage activities. The Dz209 not only showed significant intracellular reduction of BM2 gene expression in transient-expression system but also provided considerable protection against influenza B virus challenge in MDCK cells. Our findings suggest that the Dz molecule can be used as selective and effective inhibitor of viral RNA replication, and can be explored further for development of a potent therapeutic agent against influenza B virus infection.

HIV-1 VprB and C RNA Cleavage by Potent 10–23 DNAzymes That Also Cause Reversal of G2 Cell Cycle Arrest Mediated by Vpr Genes

Accessory Vpr protein of HIV-1 is known to influence several key cellular functions that also impacts on the HIV-1 replication cycle. Besides other activities, it alone causes cell cycle arrest at the G2 phase and thus potentially contribute to the overall pathology. We designed several 10-23 catalytic motifs containing DNAzymes (Dzs) against the full-length Vpr gene from subtype B and checked its activity against VprC gene from one of the Indian HIV-1 isolates. Among several Dzs that showed sequence-specific cleavage activities, Dz-94 was very potent and equally efficient in its ability to cleave full-length VprB and C RNA to completion under standard conditions of cleavage. Although Dz-90 target sequence was fully conserved between VprB and C genes, it was more effective on latter genes, suggesting that spatial structures of RNA at other regions of Vpr can also influence the cleavage activity for this Dz. HIV-1 VprB and C encoding genes under the powerful CMV promoter, when cotransfected into mammalian cells with Dz-94, a potent intracellular inhibition, was observed, which also resulted in reversing the G2 cell cycle arrest mediated by VprB and C proteins. Thus, Dz-94 could potentially be developed to prevent Vpr-mediated cytopathic effects caused by HIV-1 subtype B and C isolates.

Potent knock down of HIV-1 replication by targeting HIV-1 Tat/Rev RNA sequences synergistically with catalytic RNA and DNA

Ribozymes (Rzs) and DNA-enzymes (Dzs) possess the ability to prevent gene expression by cleaving target RNA in a catalytic and sequence-specific manner. Although Rzs or Dzs have been used earlier for HIV-1 gene suppression, the present study explored the possibility of using catalytic RNA and DNA simultaneously in a synergistic manner with the hope that this novel approach will allow more potent inhibition for a longer duration. In order to achieve long-term inhibition of HIV-1 replication, a novel non-GUX hammerhead Rz was designed by standard recombinant DNA technology and cloned it under the powerful CMV promoter containing expression vector. A 10-23 catalytic motif containing Dz that was targeted against the conserved second exon of HIV-1 Tat/Rev region was also assembled. Both Rz and Dz possessed sequence-specific cleavage activities individually and simultaneously cleaved target RNA in a synergistic manner under the same in vitro cleavage conditions. These catalytic molecules inhibited HIV-1 replication in macrophages individually and exhibited potent inhibitory effects when used in combination. The combination strategy described here can be widely used against any target RNA to achieve more effective gene inhibition that exploits the simultaneous sequence-specific cleavage potentials of catalytic RNA and DNA.

Sequence-Specific Cleavage of Hepatitis X RNA incisandtransby Novel Monotarget and Multitarget Hammerhead Motif-Containing Ribozymes

We constructed two monoribozymes and a diribozyme against the conserved region of the X RNA of hepatitis B virus (HBV). All the ribozymes (Rzs) possessed sequence-specific cleavage activities under standard and simulated physiologic conditions. Specific cleavage was also obtained when the same Rzs were placed in cis configuration with respect to X gene in multiple combinations. Rz-expressing cells were able to specifically interfere with the functional expression of X RNA and protein production in a liver-specific cell line HepG2. Potential applications of these novel Rzs are discussed.

Sequence-specific cleavage activities of DNA enzymes targeted against HIV-1 Gag and Nef regions.

The genome of HIV-1 is known to accumulate nucleotide changes throughout the course of disease that result in generation of escape mutants. Therefore, any nucleic acid-based antiviral approach should be targeted against multiple regions of the HIV-1 genome that might significantly delay the appearance of such mutants. We designed several DNA enzymes against the most conserved p24 Gag and the Nef regions in the HIV-1 genome. Sequence-specific cleavage activity was observed for all the DNA enzymes tested. Gag DNA enzyme, which cleaved the target RNA more efficiently in the presence of low levels or physiologic levels of Mg(2+), interfered more effectively with HIV-1 gene expression in virus challenge experiments. The two Nef DNA enzymes, as observed with Gag DNA enzymes, showed significant variation in their cleavage activities in the presence of varying concentration of Mg(2+) and, as expected, did not interfere with the replication of a laboratory-adapted HIV-1 isolate under in vitro culture conditions. The Gag DNA enzymes could be exploited in combination with other promising antiviral approaches.

Inhibition of HIV-1 gene expression by novel DNA enzymes targeted to cleave HIV-1 TAR RNA: potential effectiveness against all HIV-1 isolates

Nucleic acid-based antiviral approaches have been tried against multiple HIV-1 genes with the purpose of down-regulating its replication. A unique stem–loop structure called TAR is present at the 5′-end of all HIV-1 transcripts; Tat and other cellular proteins bind to TAR and thus govern transcription. Therefore, HIV-1 TAR is an attractive target against which various antiviral approaches could be tried. We screened several DNA enzymes (Dz's) containing the 10-23 catalytic motif and a single Dz containing the 8-17 catalytic motif against the HIV-1 TAR RNA. Dz's directed against the predicted single-stranded bulge regions showed sequence-specific cleavage activities. One of the two Dz's, namely Dz-475, showed moderate cleavage activity in complete absence of Mg2+. Addition of unrelated sequences at the 5′-end of the HIV-1 TAR RNA rendered it susceptible to four additional Dz-mediated cleavages. Both Dz's (470 and 475) showed significant intracellular reduction of HIV-1 gene expression. Dz-475-treated cells showed significant protection against T-tropic and macrophage-tropic HIV-1 challenge. Dz-475-transfected T-lymphocytes, human PBMCs, or chronically infected cell lines showed marked viral resistance. Unique features of this antiviral strategy with respect to HIV-1 gene inhibition are discussed.

Interaction of the resolving enzyme YDC2 with the four-way DNA junction.

Holliday junctions (four-way DNA junctions), formed during homologous recombination, are bound and resolved by junction-specific endonucleases to yield recombinant duplex DNA products. The junction-resolving enzymes are a structurally diverse class of proteins that nevertheless have many properties in common; in particular a high structure specificity for binding and metal-dependent, (frequently) sequence-specific cleavage activity. In Saccharomyces cerevisiae, the enzyme CCE1 is necessary for the resolution of recombining mitochondrial genomes, and in Schizosaccharomyces pombe the homologous protein YDC2 is thought to have a similar function. We have generated an inactive mutant of YDC2, D226N, that retains structure-specific junction binding and have analysed the interaction of this protein with the four-way DNA junction. YDC2 binds the four-way junction in two specific complexes (I and II), unfolding the stacked X-structure into a conformation where the arms extend to the four corners of a square. This structure is reminiscent of that of the free junction in the absence of metal ions and of the structures imposed on the Holliday junction by CCE1 and RuvA. DNase I probing reveals footprints specific for complexes I and II which extend from the junction centre on all four arms. No protection is observed with the small, hydrophobic probe DMS.