Simultaneous formation of specific covalent linkages at nucleotides in given DNA sequences demand distinct orthogonal reactivity of DNA modification agents. Such highly specific reactions require well-balanced reactivity and affinity of the DNA modification agents. Conjugation of a sequence-specific DNA binding zinc finger protein and a self-ligating protein tag provides a modular adaptor that expedites formation of a covalent bond between the protein tag and a substrate-modified nucleotide at a specific DNA sequence. The modular adaptor stably locates a protein of interest fused to it at the target position on DNA scaffold in its functional form. Modular adaptors with orthogonal selectivity and fast reaction kinetics to specific DNA sequences enable site-specific location of different protein molecules simultaneously. Three different modular adaptors consisting of zinc finger proteins with distinct DNA sequence specificities and self-ligating protein tags with different substrate specificities achieved orthogonal covalent bond formation at respective sequences on the same DNA scaffold with an overall coassembly yield over 90%. Application of this unique set of orthogonal modular adaptors enabled construction of a cascade reaction of three enzymes from xylose metabolic pathway on DNA scaffold.

We report the construction of an artificial enzyme cascade based on the xylose metabolic pathway. Two enzymes, xylose reductase and xylitol dehydrogenase, were assembled at specific locations on DNA origami by using DNA-binding protein adaptors with systematic variations in the interenzyme distances and defined numbers of enzyme molecules. The reaction system, which localized the two enzymes in close proximity to facilitate transport of reaction intermediates, resulted in significantly higher yields of the conversion of xylose into xylulose through the intermediate xylitol with recycling of the cofactor NADH. Analysis of the initial reaction rate, regenerated amount of NADH, and simulation of the intermediates’ diffusion indicated that the intermediates diffused to the second enzyme by Brownian motion. The efficiency of the cascade reaction with the bimolecular transport of xylitol and NAD+ likely depends more on the interenzyme distance than that of the cascade reaction with unimolecular transport between two enzymes.

A modular adaptor consisting of a sequence-specific DNA binding zinc finger protein and a self-ligating protein-tag was developed to expedite efficient formation of a covalent linkage between an individual protein molecule and the programmed address modified with a tag-substrate on the DNA nanostructure.

Hyperphosphorylation of the microtubule-associated protein tau is believed to play a crucial role in the neurofibrillary tangles formation in Alzheimer’s disease brain. In this study, fibril formation of peptides containing the critical sequences for tau aggregation VQIINK and a plausible serine phosphorylation site of tau at its C-terminal was investigated. All the peptides formed fibrils with the typical cross-β structural core. However, stability of the fibrils was highly sensitive to the pH conditions for the phosphorylated VQIINK peptide, suggesting a regulatory role of phosphorylation for the amyloid-formation of tau.

A noncovalent RNA complex embedding an aptamer function and a fluorophore-labeled peptide affords a fluorescent ribonucleopeptide (RNP) framework for constructing fluorescent sensors. By taking an advantage of the noncovalent properties of the RNP complex, the ligand-binding and fluorescence characteristics of the fluorescent RNP can be independently tuned by taking advantage of the nature of the RNA and peptide subunits, respectively. Fluorescent sensors tailored for given measurement conditions, such as a detection wavelength and a detection concentration range for a ligand of interest can be easily identified by screening of fluorescent RNP libraries. The noncovalent configuration of a RNP becomes a disadvantage when the sensor is to be utilized at very low concentrations or when multiple sensors are applied to the same solution. Here, we report a strategy to convert a fluorescent RNP sensor in the noncovalent configuration into a covalently linked stable fluorescent RNP sensor. This covalently linked fluorescent RNP sensor enabled ligand detection at a low sensor concentration, even in cell extracts. Furthermore, application of both ATP and GTP sensors enabled simultaneous detection of ATP and GTP by monitoring each wavelength corresponding to the respective sensor. Importantly, when a fluorescein-modified ATP sensor and a pyrene-modified GTP sensor were co-incubated in the same solution, the ATP sensor responded at 535 nm only to changes in the concentration of ATP, whereas the GTP sensor detected GTP at 390 nm without any effect on the ATP sensor. Finally, simultaneous monitoring by these sensors enabled real-time measurement of adenosine deaminase enzyme reactions.

Ratiometric fluorescent sensors were constructed from RNA aptamers by generating modular ribonucleopeptide complexes. Fluorescent ribonucleopeptides containing fluorophore seminaphthorhodafluor tethered to their peptide subunit revealed a dual emission property, which permitted a ratiometric fluorescent measurement of a substrate-binding event. The strategy successfully afforded ratiometric fluorescent sensors for biologically active small ligands, tetracycline, dopamine and streptomycin.

Hyperphosphorylated forms of tau protein are the main component of paired helical filaments (PHFs) of neurofibrillary tangles in the brain of Alzheimer’s disease patients. To understand the effect of phosphorylation on the fibrillation of tau, we utilized tau-derived phosphorylated peptides. The V306QIVYK311 sequence (PHF6) in the microtubule-binding domain is known to play a key role in the fibrillation of tau, and the short peptide corresponding to the PHF6 sequence forms amyloid-type fibrils similar to those generated by full-length tau. We focused on the amino acid residue located at the N-terminus of the PHF6 sequence, serine or lysine in the native isoform of tau, and synthesized the PHF6 derivative peptides with serine or lysine at the N-terminus of PHF6. Peptides phosphorylated at serine and/or tyrosine were synthesized to mimic the possible phosphorylation at these positions. The critical concentrations of the fibrillation of peptides were determined to quantitatively assess fibril stability. The peptide with the net charge of near zero tended to form stable fibrils. Interestingly, the peptide phosphorylated at the N-terminal serine residue exhibited remarkably low fibrillation propensity as compared to the peptide possessing the same net charge. Transmission electron microscopy measurements of the fibrils visualized the paired helical or straight fibers and segregated masses of the fibers or heterogeneous rodlike fibers depending on the phosphorylation status. Further analyses of the fibrils by the X-ray fiber diffraction method and Fourier transform infrared spectroscopic measurements indicated that all the peptides shared a common cross-β structure. In addition, the phosphoserine-containing peptides showed the characteristics of β-sandwiches that could interact with both faces of the β-sheet. On the basis of these observations, possible protofilament models with four β-sheets were constructed to consider the positional effects of the serine and/or tyrosine phosphorylations. The electrostatic intersheet interaction between phosphate groups and the amino group of lysine enhanced the lateral association between β-sheets to compensate for the excess charge. In addition to the previously postulated net charge of the peptide, the position of the charged residue plays a critical role in the amyloid fibrillation of tau.

A modular structure of ribonucleopeptide (RNP) affords a framework to construct macromolecular receptors and fluorescent sensors. We have isolated ATP-binding RNP with the minimum of nucleotides for ATP binding, in which the RNA consensus sequence is different from those reported for RNA aptamers against the ATP analogues. The three-dimensional structure of the substrate-binding complex of RNP was studied to understand the ATP-binding mechanism of RNP. A combination of NMR measurements, enzymatic and chemical mapping, and nucleotide mutation studies of the RNP-adenosine complex show that RNP interacts with the adenine ring of adenosine by forming a U:A:U triple with two invariant U nucleotides. The observed recognition mode for the adenine ring is different from those of RNA aptamers for ATP derivatives reported previously. The RNP-adenosine complex is folded into a particular structure by formation of the U:A:U triple and a Hoogsteen type A:U base pair. This recognition mechanism was successfully utilized to convert the substrate-binding specificity of RNP from ATP- to GTP-binding with a C+:G:C triple recognition mode.

A facile strategy of stepwise molding of a ribonucleopeptide (RNP) complex affords fluorescent RNP sensors with selective dopamine recognition. In vitro selection of a RNA-derived RNP library, a complex of the Rev peptide and its binding site Rev Responsive Element (RRE) RNA appended with random nucleotides in variable lengths, afforded RNP receptors specific for dopamine. The modular structure of the RNP receptor enables conversion of dopamine-binding RNP receptors to fluorescent dopamine sensors. Application of conditional selection schemes, such as the variation of salt concentrations and application of a counter-selection step by using a competitor ligand norepinephrine resulted in isolation of RNP receptors with defined dopamine-binding characteristics. Increasing the salt condition at the in vitro selection stage afforded RNP receptors with higher dopamine affinity, while addition of norepinephrine in the in vitro selection milieu at the counter-selection step reinforced the selectivity of RNP receptors to dopamine against norepinephrine. Thermodynamic analyses and circular dichroismic studies of the dopamine–RNP complexes suggest that the dopamine-binding RNP with higher selectivity against norepinephrine forms a pre-organized binding pocket and that the dopamine-binding RNP with higher affinity binds dopamine through the induced-fit mechanism. These results indicate that the selection condition controls the ligand-binding mechanism of RNP receptors.

Ribonucleopeptide (RNP) is a new class of scaffold for modular fluorescent sensors. We report here a short RNA motif that induces an efficient communication between the structural changes associated with the ligand-binding event of RNA aptamer and an optical response of a fluorescent RNP module. An optimized short RNA motif was used as a communication module for the rational design of modular RNP sensors. A modular combination of a GTP-binding RNA aptamer, the short RNA motif and the fluorophore-labeled RNP module afforded a fluorescent GTP sensor that retain the ligand-binding affinity of the parent aptamer.

A GTP aptamer was converted to a modular fluorescent GTP sensor by conjugation of RRE (Rev responsive element) RNA and successive complex formation with a fluorophore-modified Rev peptide. Structural changes associated with substrate binding in the RNA aptamer were successfully transduced into changes in fluorescence intensity because of the modular structure of ribonucleopeptides. A simple modular strategy involving conjugation of a fluorophore-modified ribonucleopeptide to the stem region of an RNA aptamer deduced from secondary structural information helps produce fluorescent sensors, which allow tuning of excitation and detection wavelengths through the replacement of the fluorophore at the N-terminal of the Rev peptide.Graphical abstract

Amino acid residues with aromatic side chains, such as Tyr and Phe, are known to play essential roles in forming and stabilizing the amyloid fibrils of pathogenic polypeptides by affecting their amyloid forming propensity. We have studied the amyloid-type aggregation of peptides containing non-natural amino acid derived from a core part of human pathogenic protein, tau. The hydrophobic nature of the biphenyl group and its intermolecular aromatic interactions strongly alter their amyloid formation properties.TEM images of amyloid-type fibers formed from PHF6 (left) and phenyl-substituted PHF6FPh (right). Scale bar: 100 nm. The tyrosine residue (Y) corresponds to the native Tyr-310 residue of tau.

Sequence-specific DNA binding of short peptide dimers derived from a plant basic leucine zipper protein EmBP1 was studied. A homodimer of the EmBP1 basic region peptide recognized a palindromic DNA sequence, and a heterodimer of EmBP1 and GCN4 basic region peptides targets a non-palindromic DNA sequence when a β-cyclodextrin/adamantane complex is utilized as a dimerization domain. A homodimer of the EmBP1 basic region peptide binds the native EmBP1 binding 5′-GCCACGTGGC-3′ and the native GCN4 binding 5′-ATGACGTCAT-3′ sequences with almost equal affinity in the α-helical conformation, indicating that the basic region of EmBP1 by itself has a dual recognition codes for the DNA sequences. The GCN4 basic region peptide binds 5′-ATGAC-3′ in the α-helical conformation, but it neither shows affinity nor helix formation with 5′-GCCAC-3′. Because native EmBP1 forms 100 times more stable complex with 5′-GCCACGTGGC-3′ over 5′-ATGACGTCAT-3′, our results suggest that the sequence-selectivity of native EmBP1 is dictated by the structure of leucine zipper dimerization domain including the hinge region spanning between the basic region and the leucine zipper.

•A homodimeric basic-leucine zipper protein GCN4 was applied as a new adaptor.•Orthogonal targeting by GCN4 and zif268 adaptors on DNA origami was demonstrated.•GCN4 serves as an ideal adaptor to locate homodimeric proteins on DNA origami.The addressable DNA nanostructures offer ideal platforms to construct organized assemblies of multiple protein molecules. Sequence-specific DNA binding proteins that target defined sites on DNA nanostructures would act as orthogonal adaptors to carry individual protein molecules to the programmed addresses. We have recently developed a protein-based adaptor by utilizing the sequence-specific DNA binding zinc finger protein to locate a monomeric protein of interest at specific positions on DNA origami, which serves as a molecular switchboard. We herein report a new adaptor to locate a protein dimer on the DNA origami scaffold based on a homodimeric basic-leucine zipper protein GCN4. Specific binding of GCN4 to programmed addresses on DNA origami and orthogonal targeting by GCN4- and zinc finger protein-based adaptors to the respective addresses on DNA origami were confirmed by gel electrophoretic and AFM analyses. Furthermore, a GCN4-fused homodimeric enzyme showed even higher activity than the wild type enzyme, and exhibited avid reactivity when assembled at the specific site of DNA origami. Thus, GCN4 serves as an ideal adaptor to locate homodimeric proteins in the functional form on DNA origami.A homodimeric adaptor derived from a basic-leucine zipper protein GCN4 is orthogonally applicable with the monomeric zinc finger protein adaptor, and is capable of locating a homodimeric enzyme on DNA molecular switchboard.Download high-res image (80KB)Download full-size image

Functional screening of structurally diverse libraries consisting of proteins or nucleic acids is an effective method to obtain receptors or aptamers with unique molecular recognition characteristics. However, further modification of these selected receptors to exert a newly desired function is still a challenging task. We have constructed a library of structurally diverse ribonucleopeptides (RNPs) that are modified with a catalytic group, in which the catalytic group aligns with various orientations against the ATP binding pocket of RNA subunit. As a proof-of-principle, the screening of the constructed RNP library for the catalytic reaction of ester hydrolysis was successfully carried out. The size of both the substrate-binding RNA library and the catalytic group modified peptide library are independently expandable, and thus, the size of RNPs library could be enlarged by a combination of these two subunits. We anticipate that the library of functionalized and structurally diverse RNPs would be expanded for various other catalytic reactions.

A modular adaptor consisting of a sequence-specific DNA binding zinc finger protein and a self-ligating protein-tag was developed to expedite efficient formation of a covalent linkage between an individual protein molecule and the programmed address modified with a tag-substrate on the DNA nanostructure.

Ratiometric fluorescent sensors were constructed from RNA aptamers by generating modular ribonucleopeptide complexes. Fluorescent ribonucleopeptides containing fluorophore seminaphthorhodafluor tethered to their peptide subunit revealed a dual emission property, which permitted a ratiometric fluorescent measurement of a substrate-binding event. The strategy successfully afforded ratiometric fluorescent sensors for biologically active small ligands, tetracycline, dopamine and streptomycin.