Dual miRNases for Triple Incision of miRNA Target: Design Concept and Catalytic Performance.

Irreversible destruction of disease-associated regulatory RNA sequences offers exciting opportunities for safe and powerful therapeutic interventions against human pathophysiology. In 2017, for the first time we introduced miRNAses-miRNA-targeted conjugates of a catalytic peptide and oligonucleotide capable of cleaving an miRNA target. Herein, we report the development of Dual miRNases against oncogenic miR-21, miR-155, miR-17 and miR-18a, each containing the catalytic peptide placed in-between two short miRNA-targeted oligodeoxyribonucleotide recognition motifs. Substitution of adenines with 2-aminoadenines in the sequence of oligonucleotide "shoulders" of the Dual miRNase significantly enhanced the efficiency of hybridization with the miRNA target. It was shown that sequence-specific cleavage of the target by miRNase proceeded metal-independently at pH optimum 5.5-7.5 with an efficiency varying from 15% to 85%, depending on the miRNA sequence. A distinct advantage of the engineered nucleases is their ability to additionally recruit RNase H and cut miRNA at three different locations. Such cleavage proceeds at the central part by Dual miRNase, and at the 5'- and 3'-regions by RNase H, which significantly increases the efficiency of miRNA degradation. Due to increased activity at lowered pH Dual miRNases could provide an additional advantage in acidic tumor conditions and may be considered as efficient tumor-selective RNA-targeted therapeutic.

Influence of conjugation and other structural changes on the activity of Cu²âº based PNAzymes.

We have previously shown that PNA-neocuproine conjugates can act as artificial RNA restriction enzymes. In the present study we have additionally conjugated the PNA with different entities, such as oligoethers, peptides etc. and also constructed systems where the PNA is designed to clamp the target RNA forming a triplex. Some conjugations are detrimental for the activity while most are silent which means that conjugation can be done to alter physical properties without losing activity. Conjugation with a single oligoether close to the neocuproine does enhance the rate almost twofold compared to the system without the oligoether. The systems designed to clamp the RNA target by forming a triplex retain the activity if the added oligoT sequence is 5 PNA units or shorter and extends the arsenal of artificial RNA restriction enzymes. Changing the direction of a closing base pair, where the target RNA forms a bulge, from a GC to a CG pair enhances the rate of cleavage somewhat without compromising the selectivity, leading to the so far most efficient artificial nuclease reported.

Design, RNA cleavage and antiviral activity of new artificial ribonucleases derived from mono-, di- and tripeptides connected by linkers of different hydrophobicity.

A novel series of metal-free artificial ribonucleases (aRNases) was designed, synthesized and assessed in terms of ribonuclease activity and ability to inactivate influenza virus WSN/A33/H1N1 in vitro. The compounds were built of two short peptide fragments, which include Lys, Ser, Arg, Glu and imidazole residues in various combinations, connected by linkers of different hydrophobicity (1,12-diaminododecane or 4,9-dioxa-1,12-diaminododecane). These compounds efficiently cleaved different RNA substrates under physiological conditions at rates three to five times higher than that of artificial ribonucleases described earlier and displayed RNase A-like cleavage specificity. aRNases with the hydrophobic 1,12-diaminododecane linker displayed ribonuclease activity 3-40 times higher than aRNases with the 4,9-dioxa-1,12-diaminododecane linker. The assumed mechanism of RNA cleavage was typical for natural ribonucleases, that is, general acid-base catalysis via the formation of acid/base pairs by functional groups of amino acids present in the aRNases; the pH profile of cleavage confirmed this mechanism. The most active aRNases under study exhibited high antiviral activity and entirely inactivated influenza virus A/WSN/33/(H1N1) after a short incubation period of viral suspension under physiological conditions.

Sulfonamide-bridged nucleic acid: synthesis, high RNA selective hybridization, and high nuclease resistance.

2'-N,4'-C-(N-methylamino)sulfonylmethylene-bridged thymidine (SuNA), which has a six-membered linkage including a sulfonamide moiety, was synthesized and introduced into oligonucleotides. The oligonucleotides containing SuNA exhibited excellent nuclease resistance, a high affinity toward single-stranded RNA, and a low affinity toward single-stranded DNA compared to the natural oligonucleotide.

Rapid total synthesis of DARPin pE59 and barnase.

We report the convergent total synthesis of two proteins: DARPin pE59 and Bacillus amyloliquefaciens RNase (Barnase). Leveraging our recently developed fast-flow peptide-synthesis platform, we rapidly explored numerous conditions for the assembly of long polypeptides, and were able to mitigate common side reactions, including deletion and aspartimide products. We report general strategies for improving the synthetic quality of difficult peptide sequences with our system. High-quality protein fragments produced under optimal synthetic conditions were subjected to convergent native chemical ligation, which afforded native full-length proteins after a final desulfurization step. Both DARPin and Barnase were folded and found to be as active as their recombinant analogues.

Two human host defense ribonucleases against mycobacteria, the eosinophil cationic protein (RNase 3) and RNase 7.

There is an urgent need to develop new agents against mycobacterial infections, such as tuberculosis and other respiratory tract or skin affections. In this study, we have tested two human antimicrobial RNases against mycobacteria. RNase 3, also called the eosinophil cationic protein, and RNase 7 are two small cationic proteins secreted by innate cells during host defense. Both proteins are induced upon infection displaying a wide range of antipathogen activities. In particular, they are released by leukocytes and epithelial cells, contributing to tissue protection. Here, the two RNases have been proven effective against Mycobacterium vaccae at a low micromolar level. High bactericidal activity correlated with their bacterial membrane depolarization and permeabilization activities. Further analysis on both protein-derived peptides identified for RNase 3 an N-terminus fragment that is even more active than the parental protein. Also, a potent bacterial agglutinating activity was unique to RNase 3 and its derived peptide. The particular biophysical properties of the RNase 3 active peptide are envisaged as a suitable reference for the development of novel antimycobacterial drugs. The results support the contribution of secreted RNases to the host immune response against mycobacteria.

Chemoenzymatic synthesis and lectin recognition of a selectively fluorinated glycoprotein.

A chemoenzymatic glycosylation remodeling method for the synthesis of selectively fluorinated glycoproteins is described. The method consists of chemical synthesis of a fluoroglycan oxazoline and its use as donor substrate for endoglycosidase (ENGase)-catalyzed transglycosylation to a GlcNAc-protein to form a homogeneous fluoroglycoprotein. The approach was exemplified by the synthesis of fluorinated glycoforms of ribonuclease B (RNase B). An interesting finding was that fluorination at the C-6 of the 6-branched mannose moiety in the Man3GlcNAc core resulted in significantly enhanced reactivity of the substrate in enzymatic transglycosylation. A structural analysis suggests that the enhancement in reactivity may come from favorable hydrophobic interactions between the fluorine and a tyrosine residue in the catalytic site of the enzyme (Endo-A). SPR analysis of the binding of the fluorinated glycoproteins with lectin concanavalin A (con A) revealed the importance of the 6-hydroxyl group on the α-1,6-branched mannose moiety in con A recognition. The present study establishes a facile method for preparation of selectively fluorinated glycoproteins that can serve as valuable probes for elucidating specific carbohydrate-protein interactions.

Convergent synthesis of homogeneous Glc1Man9GlcNAc2-protein and derivatives as ligands of molecular chaperones in protein quality control.

A detailed understanding of the molecular mechanism of chaperone-assisted protein quality control is often hampered by the lack of well-defined homogeneous glycoprotein probes. We describe here a highly convergent chemoenzymatic synthesis of the monoglucosylated glycoforms of bovine ribonuclease (RNase) as specific ligands of lectin-like chaperones calnexin (CNX) and calreticulin (CRT) that are known to recognize the monoglucosylated high-mannose oligosaccharide component of glycoproteins in protein folding. The synthesis of a selectively modified glycoform Gal(1)Glc(1)Man(9)GlcNAc(2)-RNase was accomplished by chemical synthesis of a large N-glycan oxazoline and its subsequent enzymatic ligation to GlcNAc-RNase under the catalysis of a glycosynthase. Selective removal of the terminal galactose by a ß-galactosidase gave the Glc(1)Man(9)GlcNAc(2)-RNase glycoform in excellent yield. CD spectroscopic analysis and RNA-hydrolyzing assay indicated that the synthetic RNase glycoforms maintained essentially the same global conformations and were fully active as the natural bovine ribonuclease B. SPR binding studies revealed that the Glc(1)Man(9)GlcNAc(2)-RNase had high affinity to lectin CRT, while the synthetic Man(9)GlcNAc(2)-RNase glycoform and natural RNase B did not show CRT-binding activity. These results confirmed the essential role of the glucose moiety in the chaperone molecular recognition. Interestingly, the galactose-masked glycoform Gal(1)Glc(1)Man(9)GlcNAc(2)-RNase also showed significant affinity to lectin CRT, suggesting that a galactose ß-1,4-linked to the key glucose moiety does not significantly block the lectin binding. These synthetic homogeneous glycoprotein probes should be valuable for a detailed mechanistic study on how molecular chaperones work in concert to distinguish between misfolded and folded glycoproteins in the protein quality control cycle.

Inactivation of a non-enveloped RNA virus by artificial ribonucleases: honey bees and acute bee paralysis virus as a new experimental model for in vivo antiviral activity assessment.

RNA-containing viruses represent a global threat to the health and wellbeing of humans and animals. Hence, the discovery of new approaches for the design of novel vaccines and antiviral compounds attains high attention. Here we describe the potential of artificial ribonucleases (aRNases), low molecular weight compounds capable to cleave phosphodiester bonds in RNA under mild conditions, to act as antiviral compounds via destroying the genome of non-enveloped RNA viruses, and the potential of utilizing honey bee larvae and adult bees (Apis mellifera) as a novel experimental system for the screening of new antiviral compounds. Pre-incubation of an Acute bee paralysis virus (ABPV) suspension with aRNases D3-12, K-D-1 or Dp12F6 in a concentration-dependent manner increased the survival rate of bee larvae and adult bees subsequently infected with these preparations, whereas incubation of the virus with aRNases ABL3C3 or L2-3 had no effect at all. The results of RT-PCR analysis of viral RNA isolated from aRNase-treated virus particles confirmed that virus inactivation occurs via degradation of viral genomic RNA: dose-dependent inactivation of ABPV correlates well with the cleavage of viral RNA. Electron microscopy analysis revealed that the morphology of ABPV particles inactivated by aRNases remains unaffected as compared to control virus preparations. Altogether the obtained results clearly demonstrate the potential of aRNases as a new virus inactivation agents and bee larvae/ABPV as a new in vivo system for the screening of antiviral compounds.

The linear assembly of a pure glycoenzyme.

A labor of love: The synthesis of an active pure enzyme (RNase) glycoform by the native chemical ligation of a (glyco)peptide and peptide thioester fragments required numerous painstaking steps: the strategy was optimized, active inteins accessed, redox conditions fine-tuned, and glycoamino acid building blocks synthesized.

Design and synthesis of peptide-oligonucleotide conjugates as potential artificial ribonucleases.

A series of peptide-oligonucleotide conjugates was synthesized. The peptide fragment was attached to the middle part of DNA sequence. For this purpose the new amidophosphite dC with linker at C5-position was synthesized.

RNA cleavage by 2,9-diamino-1,10-phenanthroline PNA conjugates.

We report on the synthesis of 2,9-diamino-1,10-phenanthroline PNA conjugates as well as on their action in cleavage of a target RNA. Synthesis of the PNA conjugates are performed on solid support and the phenanthroline derivative is conjugated either to the amino-end or to a centrally positioned diaminopropionic acid in the PNA via a urea linker. Cleavage of the target RNA is achieved and compared to cleavage with the corresponding 2,9-dimethyl-1,10-phenanthroline and glycine conjugates.

[Polycationic catalysts for phosphodiester bond cleavage on the basis of 1,4-diazabicyclo[2.2.2]octane].

A number of tetracationic compounds capable of phosphodiester bond cleavage within a 21 -membered ribooligonucleotide were designed and synthesized. The artificial ribonucleases represent two residues of quaternized 1,4-diazabicyclo[2.2.2]octane bearing alkyl substituents of various lengths and connected with a rigid linker. The efficiency of cleavage of phosphodiester bonds in an RNA target depends on the linker structure and the length of alkyl substituent.

Combinatorial experimental protocols for Erbicin-derived immunoagents and Herceptin.

Erbicin is a human anti-ErbB2 single-chain antibody fragment with high affinity and selectivity for ErbB2-positive cancer cells. Two anti-ErbB2 immunoconjugates, called Erb-hRNase and Erb-hcAb, have been prepared and found to be selectively cytotoxic on ErbB2-positive cancer cells in vitro and vivo. In Erb-hRNase, Erbicin is linked to a human RNase and in Erb-hcAb it is linked to the key structural and functional regions of a human IgG. Herceptin is an anti-ErbB2 humanised antibody successfully used in the immunotherapy of breast cancer. We report here that the Erbicin-derived immunoagents target on breast cancer cells an ErbB2 epitope different than that of Herceptin. This finding led us to verify the effects of Herceptin on breast cancer cells when it was used in combination with the Erbicin-derived immunoagents. The results indicated that in combination experiments the antitumour action of Herceptin and that of the novel agents were significantly increased in an additive fashion. An inspection of the mechanism of action of Erb-hRNase or Erb-hcAb combined with Herceptin provided evidence that the antibody combinations engendered an increased downregulation of the ErbB2 receptor, and led to an enhanced apoptotic cell death.

Di(azacrown) conjugates of 2'-O-methyl oligoribonucleotides as sequence-selective artificial ribonucleases.

Functionalized 2'-O-methyl oligoribonucleotides bearing two 3-(3-hydroxypropyl)-1,5,9-triazacyclododecane ligands attached via a phosphodiester linkage to a single non-nucleosidic building block have been prepared on a solid-support by conventional phosphoramidite chemistry. The branching units employed for the purpose include 2,2-bis(3-hydroxypropylaminocarbonyl)propane-1,3-diol, 2-hydroxyethyl 3'-O-(2-hydroxyethyl)-beta-D-ribofuranoside, and 2-hydroxyethyl 2'-O-(2-hydroxyethyl)-beta-D-ribofuranoside. Each of these has been introduced as a phosphoramidite reagent either into the penultimate 3'-terminal site or in the middle of the oligonucleotide chain. The dinuclear Zn2+ complexes of these conjugates have been shown to exhibit enhanced catalytic activity over their monofunctionalized counterpart, the 3'-terminal conjugate derived from 2-hydroxyethyl 3'-O-(2-hydroxyethyl)-beta-D-ribofuranoside being the most efficient cleaving agent. This conjugate cleaves an oligoribonucleotide target at a single phosphodiester bond and shows turnover and 1000-fold cleaving activity compared to the free monomeric Zn2+ chelate of 1,5,9-triazacyclododecane.

Design and synthesis of metal-free artificial ribonucleases.

The present review is aimed at giving a general overview of our results in the field of designing and synthesizing simple peptide-like molecules that mimic structural and functional aspects of natural ribonucleases, as well as designing oligonucleotide-based artificial ribonucleases.

Synthesis and ribonuclease activity of oligonucleotides with N3-terpyridine x Cu(II)-linked thymine residues.

In previous studies of RNA cleavage, we prepared 2'-O-methyloligonucleotide-terpyridine x Cu(II) complex(es) as sequence-specific artificial ribonucleases, in which the terpyridine group was attached to the nucleoside-sugar moiety. We found that the antisense 2'-O-methyloligomer with two terpyridine x Cu(II) complexes at contiguous internal sites was most active, and precisely cleaved the target RNA oligomer at the predetermined site. In order to explore more efficient cleavers, we have constructed a structurally similar 18-mer, but with two modified thymidine residues, in which the complex was attached to the base-N(3) position by a hydroxyethyl linker. The reaction of the target RNA 24-mer with a 10-fold excess of the new agent, in the presence of Cu(II) ions, and at pH 7.5 and 37 degrees C, revealed that the substrate was almost completely cleaved after 10 hr, and that the cleavage occurred at two contiguous sites. The activity of the new cleaver was more than half of that of the previous cleaver.

Artificial ribonucleases.

Mimicking the action of enzymes by simpler and more robust man-made catalysts has long inspired bioorganic chemists. During the past decade, mimics for RNA-cleaving enzymes, ribonucleases, or, more precisely, mimics of ribozymes that cleave RNA in sequence-selective rather than base-selective manner, have received special attention. These artificial ribonucleases are typically oligonucleotides (or their structural analogs) that bear a catalytically active conjugate group and catalyze sequence-selective hydrolysis of RNA phosphodiester bonds.

Hydrolysis of plasmid DNA and RNA by amino alkyl naphthalimide as metal-free artificial nuclease.

A strategy of dimethylamino alkyldiimide conjugated with an intercalator of naphthalimide for hydrolysis of DNA was suggested and evaluated. 4 can hydrolyze 4 kb plasmid DNA into 2 kb fragments with GC and GG selectivity, which represents a novel example of sequence- or site-selective metal-free DNA artificial nuclease. Results also show it could hydrolyze RNA efficiently.