Recognition of mismatched sites in double-stranded DNA by a pair of partially noncomplementary peptide nucleic acids.

We have successfully achieved efficient recognition of mismatched sites in double-stranded DNA through the formation of an invasion complex by using partially noncomplementary peptide nucleic acids (PNAs). Owing to mismatches between 2 PNAs used for invasion, the undesired PNA/PNA duplex, which inhibits invasion complex formation, was destabilized. This approach overcame an inherent challenge in PNA invasion, in particular, undesired PNA/PNA duplex formation resulting from PNA complementarity, thereby enhancing overall invasion efficiency.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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).

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).

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.

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.

Clear-cut observation of PNA invasion using nanomechanical DNA origami devices.

Invasive binding event of PNA into DNA duplex was clearly observed both by atomic force microscope (AFM) imaging and electrophoretic mobility shift assay (EMSA) with the aid of nanomechanical DNA origami devices as 'single-molecule' visual probes, showing their potential as universal platform for the analysis of PNA invasion.

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.

Manipulation of single-stranded DNA by using an artificial site-selective DNA cutter composed of cerium(IV)/EDTA and phosphonate-oligonucleotide conjugates.

An artificial site-selective DNA cutter to hydrolyze single-stranded DNA at a desired site was prepared from Ce(IV)/ethylenediamintetraacetic acid (EDTA) and two ethylenediamine-N,N,N',N'-tetrakis(methylenephosphonic acid)-oligonucleotide conjugates. By using this cutter, the sense strand of a blue fluorescent protein (BFP) gene was selectively cut at a predetermined site in the chromophore-coding region. The upstream fragment obtained by the site-selective scission was ligated with the downstream fragment of the closely related green fluorescent protein (GFP) gene so that the 5'- and 3'-end portions of the chromophore came from the BFP fragment and the GFP fragment, respectively.

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.

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.

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.

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.

Handy and prompt DNA separation using PNA with internal disulfide bond.

PNA (peptide nucleic acid) is a DNA analogue which has a peptide backbone. PNA is very useful as a recognition probe because of its immensely high affinity to DNA. In this study, we used PNA as a DNA separation tool.

Introduction of linkers into PNA for versatile applications.

PNA (peptide nucleic acid) is widely used as a DNA recognition tool. In this study, we introduced linkers in the middle of PNAs to expand their applicability. By means of Tm measurement and gel-shift assay, it was confirmed that these PNA can recognize double stranded DNA through efficient invasion.

Artificial restriction DNA cutter for site-selective scission of double-stranded DNA with tunable scission site and specificity.

The artificial restriction DNA cutter (ARCUT) method to cut double-stranded DNA at designated sites has been developed. The strategy at the base of this approach, which does not rely on restriction enzymes, is comprised of two stages: (i) two strands of pseudo-complementary peptide nucleic acid (pcPNA) anneal with DNA to form 'hot spots' for scission, and (ii) the Ce(IV)/EDTA complex acts as catalytic molecular scissors. The scission fragments, obtained by hydrolyzing target phosphodiester linkages, can be connected with foreign DNA using DNA ligase.

Solid-phase synthesis of pseudo-complementary peptide nucleic acids.

Pseudo-complementary peptide nucleic acid (pcPNA) is a DNA analog in which modified DNA bases 2,6-diaminopurine (D) and 2-thiouracil (U(s)) 'decorate' a poly[N-(2-aminoethyl)glycine] backbone, together with guanine (G) and cytosine (C). One of the most significant characteristics of pcPNA is its ability to effect double-duplex invasion of predetermined DNA sites inducing various changes in the biological and the physicochemical properties of the DNA. This protocol describes solid-phase synthesis of pcPNA.