A Compound I Mimic Reveals the Transient Active Species of a Cytochrome P450 Enzyme: Insight into the Stereoselectivity of P450-Catalysed Oxidations.

Catching the structure of cytochrome P450 enzymes in flagrante is crucial for the development of P450 biocatalysts, as most structures collected are found trapped in a precatalytic conformation. At the heart of P450 catalysis lies Cpd I, a short-lived, highly reactive intermediate, whose recalcitrant nature has thwarted most attempts at capturing catalytically relevant poses of P450s. We report the crystal structure of P450BM3 mimicking the state in the precise moment preceding epoxidation, which is in perfect agreement with the experimentally observed stereoselectivity. This structure was attained by incorporation of the stable Cpd I mimic oxomolybdenum mesoporphyrin IX into P450BM3 in the presence of styrene. The orientation of styrene to the Mo-oxo species in the crystal structures sheds light onto the dynamics involved in the rotation of styrene to present its vinyl group to Cpd I. This method serves as a powerful tool for predicting and modelling the stereoselectivity of P450 reactions.

Hoodwinking Cytochrome P450BM3 into Hydroxylating Non-Native Substrates by Exploiting Its Substrate Misrecognition.

Bacterial cytochrome P450s (P450s) are at the focus of attention as potential biocatalysts for applications in green synthetic chemistry, as they possess high activity for the hydroxylation of inert substrate C-H bonds. The high activity of bacterial P450s, such as P450BM3, is chiefly due to their high substrate specificity, and consequently, the catalytic activity of P450BM3 toward non-native substrates is very low, limiting the utility of bacterial P450s as biocatalysts. To enable oxidation of non-native substrates by P450BM3 without any mutagenesis, we have developed a series of "decoy molecules", inert dummy substrates, with structures that resemble those of the native substrates. Decoy molecules fool P450BM3 into generating the active species, so-called Compound I, enabling the catalytic oxidation of non-native substrates other than fatty acids. Perfluorinated carboxylic acids (PFCs) serve as decoy molecules to initiate the activation of molecular oxygen in the same manner as long-alkyl-chain fatty acids, due to their structural similarity, and induce the generation of Compound I, but, unlike the native substrates, PFCs are not oxidizable by Compound I, allowing the hydroxylation of non-native substrates, such as gaseous alkanes and benzene. The catalytic activity for non-native substrate hydroxylation was significantly enhanced by employing second generation decoy molecules, PFCs modified with amino acids (PFC-amino acids). Cocrystals of P450BM3 with PFC9-Trp revealed clear electron density in the fatty-acid-binding channel that was readily assigned to PFC9-Trp. The alkyl chain terminus of PFC9-Trp does not reach the active site owing to multiple hydrogen bonding interactions between the carboxyl and carbonyl groups of PFC9-Trp and amino acids located at the entrance of the substrate binding channel of P450BM3 that fix it in place. The remaining space above the heme after binding of PFC9-Trp can be utilized to accommodate non-native substrates. Further developments revealed that third generation decoy molecules, N-acyl amino acids, such as pelargonoyl-l-phenylalanine (C9-Phe), can serve as decoy molecules, indicating that the rationale "fluorination is required for decoy molecule function" can be safely discarded. Diverse carboxylic acids including dipeptides could now be exploited as building blocks, and a library of decoy molecules possessing diverse structures was prepared. Among the third-generation decoy molecules examined N-enanthyl-l-proline modified with l-phenylalanine (C7-Pro-Phe) afforded the maximum turnover rate for benzene hydroxylation. The structural diversity of third-generation decoy molecules was also utilized to control the stereoselectivity of hydroxylation for the benzylic hydroxylation of Indane, showing that decoy molecules can alter stereoselectivity. As both the catalytic activity and enantioselectivity are dependent upon the structure of the decoy molecules, their design allows us to regulate reactions catalyzed by wild-type enzymes. Furthermore, decoy molecules can also activate intracellular P450BM3, allowing the use of E. coli expressing wild-type P450BM3 as an efficient whole-cell bioreactor. It should be noted that Mn-substituted full-length P450BM3 (Mn-P450BM3) is also active for the hydroxylation of propane in which the regioselectivity diverged from that of Fe-P450BM3. The results summarized in this Account represent good examples of how the reactive properties of P450BM3 can be controlled for the monooxygenation of non-native substrates in vitro as well as in vivo to expand the potential of P450BM3.

Peptide Nucleic Acid Conjugated with Ruthenium-Complex Stabilizing Double-Duplex Invasion Complex Even under Physiological Conditions.

Peptide nucleic acid (PNA) can form a stable duplex with DNA, and, accordingly, directly recognize double-stranded DNA through the formation of a double-duplex invasion complex, wherein a pair of complementary PNA strands form two PNA/DNA duplexes. Because invasion does not require prior denaturation of DNA, PNA holds great potential for in cellulo or in vivo applications. To broaden the applicability of PNA invasion, we developed a new conjugate of PNA with a ruthenium complex. This Ru-PNA conjugate exhibits higher DNA-binding affinity, which results in enhanced invasion efficiency, even under physiological conditions.

Promotion of double-duplex invasion of peptide nucleic acids through conjugation with nuclear localization signal peptide.

Pseudo-complementary peptide nucleic acid (pcPNA), as one of the most widely used synthetic DNA analogues, invades double-stranded DNA according to Watson-Crick rules to form invasion complexes. This unique mode of DNA recognition induces structural changes at the invasion site and can be used for a range of applications. In this paper, pcPNA is conjugated with a nuclear localization signal (NLS) peptide, and its invading activity is notably promoted both thermodynamically and kinetically. Thus, the double-duplex invasion complex is formed promptly at low pcPNA concentrations under high salt conditions, where the invasion otherwise never occurs. Furthermore, NLS-modified pcPNA is successfully employed for site-selective DNA scission, and the targeted DNA is selectively cleaved under conditions that are not conducive for DNA cutters using unmodified pcPNAs. This strategy of pcPNA modification is expected to be advantageous and promising for a range of in vitro and in vivo applications.

ss-siRNAs allele selectively inhibit ataxin-3 expression: multiple mechanisms for an alternative gene silencing strategy.

Single-stranded silencing RNAs (ss-siRNAs) provide an alternative approach to gene silencing. ss-siRNAs combine the simplicity and favorable biodistribution of antisense oligonucleotides with robust silencing through RNA interference (RNAi). Previous studies reported potent and allele-selective inhibition of human huntingtin expression by ss-siRNAs that target the expanded CAG repeats within the mutant allele. Mutant ataxin-3, the genetic cause of Machado-Joseph Disease, also contains an expanded CAG repeat. We demonstrate here that ss-siRNAs are allele-selective inhibitors of ataxin-3 expression and then redesign ss-siRNAs to optimize their selectivity. We find that both RNAi-related and non-RNAi-related mechanisms affect gene expression by either blocking translation or affecting alternative splicing. These results have four broad implications: (i) ss-siRNAs will not always behave similarly to analogous RNA duplexes; (ii) the sequences surrounding CAG repeats affect allele-selectivity of anti-CAG oligonucleotides; (iii) ss-siRNAs can function through multiple mechanisms and; and (iv) it is possible to use chemical modification to optimize ss-siRNA properties and improve their potential for drug discovery.

Heme-substituted protein assembly bridged by synthetic porphyrin: achieving controlled configuration while maintaining rotational freedom.

The use of biological host-guest interactions, specifically the binding of hemoprotein to heme, has attracted significant research interest in the design of artificial protein assemblies. However, because of the inherent flexibility of the propionic acid group of heme, it is difficult to control the positioning and orientation of the protein unit and to construct well-ordered structures. Herein, we report a heme-substituted protein dimer composed of the native hemoprotein HasA, which accommodates a tetraphenylporphyrin bearing an additional metal coordination site. The specific binding of the tetraphenylporphyrin with an additional metal coordination site that protrudes in a fixed direction confines the configuration of the dimer structure to a defined bent form. The small-angle X-ray scattering profile shows the dimer structure with a bent form and suggests dynamic rotational behavior while keeping its bent-core structure, resembling a bevel gear. This unique dimer structure demonstrates that the design of heme-substituted protein assemblies can be expanded to protein assemblies while maintaining the rotational freedom of the individual protein units.

Allele-selective inhibition of expression of huntingtin and ataxin-3 by RNA duplexes containing unlocked nucleic acid substitutions.

Unlocked nucleic acid (UNA) is an acyclic analogue of RNA that can be introduced into RNA or DNA oligonucleotides. The increased flexibility conferred by the acyclic structure fundamentally affects the strength of base pairing, creating opportunities for improved applications and new insights into molecular recognition. Here we test how UNA substitutions affect allele-selective inhibition of expression of trinucleotide repeat genes Huntingtin (HTT) and Ataxin-3 (ATX-3). We find that the either the combination of mismatched bases and UNA substitutions or UNA substitutions alone can improve potency and selectivity. Inhibition is potent, and selectivities of >40-fold for inhibiting mutant versus wild-type expression can be achieved. Surprisingly, even though UNA preserves the potential for complete base pairing, the introduction of UNA substitutions at central positions within fully complementary duplexes leads to >19-fold selectivity. Like mismatched bases, the introduction of central UNA bases disrupts the potential for cleavage of substrate by argonaute 2 (AGO2) during gene silencing. UNA-substituted duplexes are as effective as other strategies for allele-selective silencing of trinucleotide repeat disease genes. Modulation of AGO2 activity by the introduction of UNA substitutions demonstrates that backbone flexibility is as important as base pairing for catalysis of fully complementary duplex substrates. UNA can be used to tailor RNA silencing for optimal properties and allele-selective action.

PNA-NLS conjugates as single-molecular activators of target sites in double-stranded DNA for site-selective scission.

Artificial DNA cutters have been developed by us in our previous studies by combining two strands of pseudo-complementary peptide nucleic acid (pcPNA) with Ce(IV)-EDTA-promoted hydrolysis. The pcPNAs have two modified nucleobases (2,6-diaminopurine and 2-thiouracil) instead of conventional A and T, and can invade double-stranded DNA to activate the target site for the scission. This system has been applied to site-selective scissions of plasmid, λ-phage, E. coli genomic DNA, and human genomic DNA. Here, we have reported a still simpler and more convenient DNA cutter obtained by conjugating peptide nucleic acid (PNA) with a nuclear localization signal (NLS) peptide. This new DNA cutter requires only one PNA strand (instead of two) bearing conventional (non-pseudo-complementary) nucleobases. This PNA-NLS conjugate effectively activated the target site in double-stranded DNA and induced site-selective scission by Ce(IV)-EDTA. The complex formation between the conjugate and DNA was concretely evidenced by spectroscopic results based on time-resolved fluorescence. The target scission site of this new system was straightforwardly determined by the Watson-Crick base pairing rule, and mismatched sequences were clearly discriminated. Importantly, even highly GC-rich regions, which are difficult to be targeted by a previous strategy using pcPNA, were successfully targeted. All these features of the present DNA cutter make it promising for various future applications.

Artificial DNA cutters for DNA manipulation and genome engineering.

This tutorial review provides recent developments in artificial cutters for site-selective scission of DNA with the focus on chemistry-based DNA cutters. They are useful tools for molecular biology and biotechnology, since their site-selectivity of scission is much higher than that of naturally occurring restriction enzymes and also their scission site is freely chosen. In order to prepare these cutters, a DNA-cutting molecule is combined with a sequence-recognizing molecule in a covalent or non-covalent way. At targeted sites in single-stranded and double-stranded DNAs, the scission occurs via either oxidative cleavage of nucleotides or hydrolysis of phosphodiester linkages. Among many successful examples, an artificial restriction DNA cutter, prepared from Ce(iv)/EDTA and pseudo-complementary peptide nucleic acid, hydrolyzed double-stranded DNA at the target site. The scission site and scission specificity are determined simply in terms of the Watson-Crick rule so that even the whole genome of human beings was selectively cut at one predetermined site. Consistently, homologous recombination in human cells was successfully promoted by this tool. For the purpose of comparison, protein-based DNA cutters (e.g., zinc finger nucleases) are also briefly described. The potential applications of these cutters and their future aspects are discussed.

C9orf72-derived arginine-rich poly-dipeptides impede phase modifiers.

Nuclear import receptors (NIRs) not only transport RNA-binding proteins (RBPs) but also modify phase transitions of RBPs by recognizing nuclear localization signals (NLSs). Toxic arginine-rich poly-dipeptides from C9orf72 interact with NIRs and cause nucleocytoplasmic transport deficit. However, the molecular basis for the toxicity of arginine-rich poly-dipeptides toward NIRs function as phase modifiers of RBPs remains unidentified. Here we show that arginine-rich poly-dipeptides impede the ability of NIRs to modify phase transitions of RBPs. Isothermal titration calorimetry and size-exclusion chromatography revealed that proline:arginine (PR) poly-dipeptides tightly bind karyopherin-ß2 (Kapß2) at 1:1 ratio. The nuclear magnetic resonances of Kapß2 perturbed by PR poly-dipeptides partially overlapped with those perturbed by the designed NLS peptide, suggesting that PR poly-dipeptides target the NLS binding site of Kapß2. The findings offer mechanistic insights into how phase transitions of RBPs are disabled in C9orf72-related neurodegeneration.

Oxidation of an oligonucleotide-bound Ce(III)/multiphosphonate complex for site-selective DNA scission.

Oligodeoxyribonucleotide conjugates of ethylenediamine-N,N,N',N'-tetrakis(methylenephosphonic acid) (EDTP) have been used to place a Ce(III)/EDTP complex in close proximity to predetermined phosphodiester linkages of a complementary target oligonucleotide. In the presence of atmospheric oxygen, the Ce(III) is oxidized into Ce(IV) which, in turn, efficiently cleaves the target phosphodiester linkage. No cleavage occurs at the other single-stranded regions, which suggests that the catalytic Ce species is strictly localized next to the target phosphodiester linkage. No decrease in the reaction rate is observed upon introduction of scavengers for hydroxyl radicals (such as DMSO or MeOH) or singlet oxygen (such as NaN(3)) to the system; this indicates that the reaction proceeds via a hydrolytic pathway. Any significant contribution by an oxidative pathway is further ruled out by the observation that nucleosides remain intact after incubation with Ce(IV)/EDTP complex for extended periods.

Chemical and enzymatic stability of amino acid derived phosphoramidates of antiviral nucleoside 5'-monophosphates bearing a biodegradable protecting group.

Ribavirin and 2'-O-methylcytidine 5'-phosphoramidates derived from L-alanine methyl ester bearing either an O-phenyl or a biodegradable O-[3-(acetyloxy)-2,2-bis(ethoxycarbonyl)propyl] or O-[3-(acetyloxymethoxy)-2,2-bis(ethoxycarbonyl)propyl] protecting group were prepared. The kinetics of the deprotection of these pro-drugs by porcine liver esterase and by a whole cell extract of human prostate carcinoma was studied by HPLC-ESI-MS/MS. The 3-(acetyloxymethoxy)-2,2-bis(ethoxycarbonyl)propyl and 3-(acetyloxy)-2,2-bis(ethoxycarbonyl)propyl groups were readily removed releasing the l-alanine methyl ester phosphoramidate nucleotide, the deprotection of the 3-(acetyloxymethoxy) derivative being approximately 20 times faster. The chemical stability of the 2'-O-methylcytidine pro-drugs was additionally determined over a pH range from 7.5 to 10.

Cationic guanine: positively charged nucleobase with improved DNA affinity inhibits self-duplex formation.

Oligonucleotides represent powerful DNA-recognition tools, but the formation of undesirable "self-duplexes" becomes more probable with increasing DNA affinity. Herein, we have developed a modified nucleobase with "self-avoiding" properties. Facile methylation of guanine yields a cationic N7-methylguanine, which suppresses the formation of self-duplexes whilst improving DNA affinity through electrostatic interaction.

Expanding the applicability of cytochrome P450s and other haemoproteins.

Cytochrome P450BM3 has long been regarded as a promising candidate for use as a biocatalyst, owing to its excellent efficiency for the hydroxylation of unactivated C-H bonds. However, because of its high substrate specificity, its possible applications have been severely limited. Consequently, various approaches have been proposed to overcome the enzyme's natural limitations, thereby expanding its substrate scope to encompass non-native substrates, evoking chemoselectivity, regioselectivity and stereoselectivity and enabling previously inaccessible chemical conversions. Herein, these approaches will be classified into three categories: (1) mutagenesis including directed evolution, (2) haem substitution with artificial cofactors and (3) use of substrate mimics, 'decoy molecules'. Herein, we highlight the representative work that has been conducted in above three categories for discussion of the future outlook of P450BM3 in green chemistry.

Introduction of disulfide bond to the main chain of PNA to switch its hybridization and invasion activity.

In order to facilitate the removal of peptide nucleic acid (PNA), when necessary, from its duplexes and invasion complexes, a disulfide bond was introduced to its main chain. The disulfide bond was readily cleaved by various reducing agents (2-mercaptoethanol, dl-dithiothreitol, and tris(2-carboxyethyl)phosphine) even when the PNA was forming a duplex with its complementary DNA. The resultant two short PNA fragments were spontaneously removed from the DNA. Double-duplex invasion complexes of two disulfide-containing PNA strands were also promptly cleaved by the reducing agents. By using this modified PNA, a desired DNA fragment was picked up from DNA mixtures, and obtained in a pure form (free from the PNA) by the reductive treatment. Importantly, this separation was achieved at low temperatures (e.g., 37 degrees C), where all the DNAs (and other biomolecules if any) should be kept intact. Strong potential of the modified PNA for various biological applications has been indicated.

Chemical modification of Ce(IV)/EDTA-based artificial restriction DNA cutter for versatile manipulation of double-stranded DNA.

A monophosphate group was attached to the terminus of pseudo-complementary peptide nucleic acid (pcPNA), and two of thus modified pcPNAs were combined with Ce(IV)/EDTA for site-selective hydrolysis of double-stranded DNA. The site-selective DNA scission was notably accelerated by this chemical modification of pcPNAs. These second-generation artificial restriction DNA cutters (ARCUTs) differentiated the target sequence so strictly that no scission occurred even when only one DNA base-pair was altered to another. By using two of the activated ARCUTs simultaneously, DNA substrate was selectively cut at two predetermined sites, and the desired fragment was clipped and cloned. The DNA scission by ARCUT was also successful even when the target site was methylated by methyltransferase and protected from the corresponding restriction enzyme. Furthermore, potentiality of ARCUT for manipulation of huge DNA has been substantiated by site-selective scission of genomic DNA of Escherichia coli (composed of 4,600,000 bp) at the target site. All these results indicate promising applications of ARCUTs for versatile purposes.

Reconstitution of full-length P450BM3 with an artificial metal complex by utilising the transpeptidase Sortase A.

Haem substitution is an effective approach to tweak the function of haemoproteins. Herein, we report a facile haem substitution method for self-sufficient cytochrome P450BM3 (CYP102A1) from Bacillus megaterium utilising the transpeptidase Sortase A from Staphylococcus aureus. We successfully constructed Mn-substituted BM3 and investigated its catalytic activity.

Enhancement of RNA cleavage activity of 10-23 DNAzyme by covalently introduced intercalator.

By introducing an intercalator through D-threoninol to the 10-23 DNAzyme at the junction between its catalytic loop and the binding arm, the RNA cleavage activity was greatly improved.

Exploring the effect of sequence length and composition on allele-selective inhibition of human huntingtin expression by single-stranded silencing RNAs.

Mutant huntingtin (HTT) protein is the cause of Huntington's disease (HD), an incurable neurological disorder. Almost all patients are heterozygous for mutant HTT and approaches that reduce levels of mutant HTT while leaving expression of wild-type HTT intact might be ideal options for therapeutic development. We have developed several allele-selective strategies for silencing HTT, including single-stranded silencing RNAs (ss-siRNAs). ss-siRNAs are oligonucleotides containing chemical modifications that permit action through the RNA interference (RNAi) pathway. Modified ss-siRNAs chosen to test the effects of varying oligomer length, lipid modification, the introduction of mismatched bases, and variation of chemical modification. We find that several modified ss-siRNA are potent and allele-selective inhibitors of HTT expression. An ss-siRNA with three mismatched bases relative to the CAG repeat was an allele-selective inhibitor of HTT expression in the HdhQ175 mouse model. Multiple allele-selective ss-siRNAs provide a wide platform of modifications to draw on for further optimization and therapeutic development. Our data provide insights into how ss-siRNAs can be modified to improve their properties and facilitate the discovery of the lead compounds necessary for further development.

Introduction of multiphosphonate ligand to peptide nucleic acid for metal ion conjugation.

Peptide nucleic acid (PNA) is one of the most widely used synthetic DNA analogs. Conjugation of functional molecules to PNA is very effective to further widen its potential applications. For this purpose, here we report the synthesis of several ligand monomers and introduced them to PNA. These ligand-modified PNAs attract cerium ion and are useful for site-selective DNA hydrolysis. It should be noted that these ligands on PNA are also effective even under the conditions of invasion complex.