NMR characterization of the structure of the intrinsically disordered region of human origin recognition complex subunit 1, hORC1, and of its interaction with G-quadruplex DNAs.

Human origin recognition complex (hORC) binds to the DNA replication origin and then initiates DNA replication. However, hORC does not exhibit DNA sequence-specificity and how hORC recognizes the replication origin on genomic DNA remains elusive. Previously, we found that hORC recognizes G-quadruplex structures potentially formed near the replication origin. Then, we showed that hORC subunit 1 (hORC1) preferentially binds to G-quadruplex DNAs using a hORC1 construct comprising residues 413 to 511 (hORC1413-511). Here, we investigate the structural characteristics of hORC1413-511 in its free and complex forms with G-quadruplex DNAs. Circular dichroism and nuclear magnetic resonance (NMR) spectroscopic studies indicated that hORC1413-511 is disordered except for a short α-helical region in both the free and complex forms. NMR chemical shift perturbation (CSP) analysis suggested that basic residues, arginines and lysines, and polar residues, serines and threonines, are involved in the G-quadruplex DNA binding. Then, this was confirmed by mutation analysis. Interestingly, CSP analysis indicated that hORC1413-511 binds to both parallel- and (3 + 1)-type G-quadruplex DNAs using the same residues, and thereby in the same manner. Our study suggests that hORC1 uses its intrinsically disordered G-quadruplex binding region to recognize parallel-type and (3 + 1)-type G-quadruplex structures at replication origin.

Complex Formation of an RNA Aptamer with a Part of HIV-1 Tat through Induction of Base Triples in Living Human Cells Proven by In-Cell NMR.

An RNA aptamer that strongly binds to a target molecule has the potential to be a nucleic acid drug inside living human cells. To investigate and improve this potential, it is critical to elucidate the structure and interaction of RNA aptamers inside living cells. We examined an RNA aptamer for HIV-1 Tat (TA), which had been found to trap Tat and repress its function in living human cells. We first used in vitro NMR to examine the interaction between TA and a part of Tat containing the binding site for trans-activation response element (TAR). It was revealed that two U-A∗U base triples are formed in TA upon binding of Tat. This was assumed to be critical for strong binding. Then, TA in complex with a part of Tat was incorporated into living human cells. The presence of two U-A∗U base triples was also revealed for the complex in living human cells by in-cell NMR. Thus, the activity of TA in living human cells was rationally elucidated by in-cell NMR.

Detection of interaction between an RNA aptamer and its target compound in living human cells using 2D in-cell NMR.

We introduced an isotopically labeled RNA aptamer for HIV-1 Tat prepared by E. coli transcription into HeLa cells. We successfully recorded the first heteronuclear 2D in-cell NMR spectra, which makes it possible to study the interaction of the RNA aptamer with argininamide in living human cells with higher resolution.

Shedding light on the base-pair opening dynamics of nucleic acids in living human cells.

Base-pair opening is a fundamental property of nucleic acids that plays important roles in biological functions. However, studying the base-pair opening dynamics inside living cells has remained challenging. Here, to determine the base-pair opening kinetics inside living human cells, the exchange rate constant ([Formula: see text]) of the imino proton with the proton of solvent water involved in hairpin and G-quadruplex (GQ) structures is determined by the in-cell NMR technique. It is deduced on determination of [Formula: see text] values that at least some G-C base pairs of the hairpin structure and all G-G base-pairs of the GQ structure open more frequently in living human cells than in vitro. It is suggested that interactions with endogenous proteins could be responsible for the increase in frequency of base-pair opening. Our studies demonstrate a difference in dynamics of nucleic acids between in-cell and in vitro conditions.

Small-angle X-ray scattering data of a guanine-rich DNA derived from the promoter region of c-MYC gene in solution.

This article presented the small-angle X-ray scattering (SAXS) data of a guanine-rich DNA derived from the promoter region of c-MYC gene (Pu22) in solution. The data is collected under the condition, where the Pu22 takes a guanine quadruplex (GQ) structure. The SAXS curve was also measured and analyzed when 18-crown-6, a chelator of K+ ions, was added to the Pu22 solution.

Detection of parallel and antiparallel DNA triplex structures in living human cells using in-cell NMR.

We introduced oligodeoxynucleotides (ODNs) that form parallel and antiparallel triplex structures in vitro into living human cells and recorded their in-cell NMR spectra. Observation of landmark signals for triplex structures proved for the first time that parallel and antiparallel triplex structures are formed in living human cells.

Investigation of the Interaction of Human Origin Recognition Complex Subunit 1 with G-Quadruplex DNAs of Human c-myc Promoter and Telomere Regions.

Origin recognition complex (ORC) binds to replication origins in eukaryotic DNAs and plays an important role in replication. Although yeast ORC is known to sequence-specifically bind to a replication origin, how human ORC recognizes a replication origin remains unknown. Previous genome-wide studies revealed that guanine (G)-rich sequences, potentially forming G-quadruplex (G4) structures, are present in most replication origins in human cells. We previously suggested that the region comprising residues 413-511 of human ORC subunit 1, hORC1413-511, binds preferentially to G-rich DNAs, which form a G4 structure in the absence of hORC1413-511. Here, we investigated the interaction of hORC1413-511 with various G-rich DNAs derived from human c-myc promoter and telomere regions. Fluorescence anisotropy revealed that hORC1413-511 binds preferentially to DNAs that have G4 structures over ones having double-stranded structures. Importantly, circular dichroism (CD) and nuclear magnetic resonance (NMR) showed that those G-rich DNAs retain the G4 structures even after binding with hORC1413-511. NMR chemical shift perturbation analyses revealed that the external G-tetrad planes of the G4 structures are the primary binding sites for hORC1413-511. The present study suggests that human ORC1 may recognize replication origins through the G4 structure.

Observation of nucleic acids inside living human cells by in-cell NMR spectroscopy.

The intracellular environment is highly crowded with biomacromolecules such as proteins and nucleic acids. Under such conditions, the structural and biophysical features of nucleic acids have been thought to be different from those in vitro. To obtain high-resolution structural information on nucleic acids in living cells, the in-cell NMR method is a unique tool. Following the first in-cell NMR measurement of nucleic acids in 2009, several interesting insights were obtained using Xenopus laevis oocytes. However, the in-cell NMR spectrum of nucleic acids in living human cells was not reported until two years ago due to the technical challenges of delivering exogenous nucleic acids. We reported the first in-cell NMR spectra of nucleic acids in living human cells in 2018, where we applied a pore-forming toxic protein, streptolysin O. The in-cell NMR measurements demonstrated that the hairpin structures of nucleic acids can be detected in living human cells. In this review article, we summarize our recent work and discuss the future prospects of the in-cell NMR technique for nucleic acids.

Recent progress of in-cell NMR of nucleic acids in living human cells.

The inside of living cells is highly crowded with biological macromolecules. It has long been considered that the properties of nucleic acids and proteins, such as their structures, dynamics, interactions, and enzymatic activities, in intracellular environments are different from those under in vitro dilute conditions. In-cell NMR is a robust and powerful method used in the direct measurement of those properties in living cells. However, until 2 years ago, in-cell NMR was limited to Xenopus laevis oocytes due to technical challenges of incorporating exogenous nucleic acids. In the last 2 years, in-cell NMR spectra of nucleic acid introduced into living human cells have been reported. By use of the in-cell NMR spectra of nucleic acids in living human cells, the formation of hairpin structures with Watson-Crick base pairs, and i-motif and G-quadruplex structures with non-Watson-Crick base pairs was demonstrated. Others investigated the mRNA-antisense drug interactions and DNA-small compound interactions. In this article, we review these studies to underscore the potential of in-cell NMR for addressing the structures, dynamics, and interactions of nucleic acids in living human cells.

RNA sequence and length contribute to RNA-induced conformational change of TLS/FUS.

Translocated in liposarcoma (TLS)/fused in sarcoma (FUS) is a multitasking DNA/RNA binding protein implicated in cancer and neurodegenerative diseases. Upon DNA damage, TLS is recruited to the upstream region of the cyclin D1 gene (CCND1) through binding to the promotor associated non-coding RNA (pncRNA) that is transcribed from and tethered at the upstream region. Binding to pncRNA is hypothesized to cause the conformational change of TLS that enables its inhibitive interaction with histone acetyltransferases and resultant repression of CCND1 expression, although no experimental proof has been obtained. Here, the closed-to-open conformational change of TLS on binding pncRNA was implied by fluorescence resonance energy transfer. A small fragment (31 nucleotides) of the full-length pncRNA (602 nucleotides) was shown to be sufficient for the conformational change of TLS. Dissection of pncRNA identified the G-rich RNA sequence that is critical for the conformational change. The length of RNA was also revealed to be critical for the conformational change. Furthermore, it was demonstrated that the conformational change of TLS is caused by another target DNA and RNA, telomeric DNA and telomeric repeat-containing RNA. The conformational change of TLS on binding target RNA/DNA is suggested to be essential for biological functions.

The anti-prion RNA aptamer R12 disrupts the Alzheimer's disease-related complex between prion and amyloid ß.

The neurodegenerative disorder Alzheimer's disease (AD) is associated with the accumulation of misfolded proteins. Some recent studies suggested that amyloid beta (Aß) forms soluble oligomers, protofibrils, and fibrils; the Aß oligomers being more toxic than the fibrils. Surprisingly, these Aß oligomers reportedly bind to prion protein (PrP), which acts as a receptor on the cell membrane, possibly resulting in AD. Thus, it is thought that compounds that can disrupt the formation of the prion-Aß oligomer complex may prevent AD. Here, we demonstrate that an anti-prion RNA aptamer, R12, inhibits the interaction of PrP with Aß. Fluorescence assaying involving thioflavin S showed that wild-type PrP, a mutant of the N-terminal half of PrP, and even fragment peptides of PrP effectively inhibit Aß fibrillization. Fluorescence anisotropy revealed that R12 is capable of binding to PrP, resulting in dissociation of PrP with Aß. Consequently, the Aß that dissociated from PrP was shown to polymerize into fibrils. These spectroscopic observations were visualized by transmission electron microscopy. This is the first demonstration of the PrP-Aß interaction being disrupted by a nucleic acid. This ability of R12 highlights its therapeutic potential for treating AD pathology.

Analysis of Interactions between Telomeric i-Motif DNA and a Cyclic Tetraoxazole Compound.

The interaction of a macrocyclic tetraoxazole compound, L2H2-4OTD (1), with two aminoalkyl side chains and telomeric i-motif, was investigated by means of electrophoretic mobility shift assay, circular dichroism spectroscopy, mass spectrometry and NMR spectroscopy analyses. The results indicate that 1 interacts with the i-motif structure at two preferred binding sites.

Selective alkylation of T-T mismatched DNA using vinyldiaminotriazine-acridine conjugate.

The alkylation of the specific higher-order nucleic acid structures is of great significance in order to control its function and gene expression. In this report, we have described the T-T mismatch selective alkylation with a vinyldiaminotriazine (VDAT)-acridine conjugate. The alkylation selectively proceeded at the N3 position of thymidine on the T-T mismatch. Interestingly, the alkylated thymidine induced base flipping of the complementary base in the duplex. In a model experiment for the alkylation of the CTG repeats DNA which causes myotonic dystrophy type 1 (DM1), the observed reaction rate for one alkylation increased in proportion to the number of T-T mismatches. In addition, we showed that primer extension reactions with DNA polymerase and transcription with RNA polymerase were stopped by the alkylation. The alkylation of the repeat DNA will efficiently work for the inhibition of replication and transcription reactions. These functions of the VDAT-acridine conjugate would be useful as a new biochemical tool for the study of CTG repeats and may provide a new strategy for the molecular therapy of DM1.

The first successful observation of in-cell NMR signals of DNA and RNA in living human cells.

In order to understand intracellular biological events, information on the structure, dynamics and interaction of proteins and nucleic acids in living cells is of crucial importance. In-cell NMR is a promising method to obtain this information. Although NMR signals of proteins in human cells have been reported, those of nucleic acids were reported only in Xenopus laevis oocytes, i.e., not in human cells. Here, DNA and RNA were introduced into human cells by means of pore formation by bacterial toxin streptolysin O and subsequent resealing. Then, NMR signals of DNA and RNA were successfully observed for the first time in living human cells. The observed signals directly suggested the formation of DNA and RNA hairpin structures in living human cells.

Development of an RNA aptamer that acquires binding capacity against HIV-1 Tat protein via G-quadruplex formation in response to potassium ions.

For the development of K+-responsive RNA aptamers, we proposed a new general strategy that makes use of a G-quadruplex formation in response to K+. This is the first report of developing an RNA aptamer that demonstrates ON/OFF switching of its target-binding activity by sensing the addition/removal of K+.

K(+)-Responsive off-to-on switching of hammerhead ribozyme through dual G-quadruplex formation requiring no heating and cooling treatment.

Functional RNAs that switch their activities in response to K(+) may sense the intracellular (100 mM) and extracellular (5 mM) K(+) concentrations and regulate their functions accordingly. Previously, we developed a quadruplex hammerhead ribozyme (QHR) whose conformational change, from a duplex to a G-quadruplex, triggered by K(+) results in expression of the activity. However, this QHR required heating and cooling treatment (annealing) to induce the K(+)-responsive conformational change and activity. Here, we developed a new quadruplex hammerhead ribozyme (QHR) system that does not require annealing to induce the K(+)-responsive conformational change and activity. This system is composed of QHR and a G-quadruplex-forming complementary DNA strand (QCS). In the absence of K(+), QCS formed a duplex with QHR, which suppressed the residual activity. Upon elevation of the K(+) concentration, QCS dissociated from QHR was trapped in a G-quadruplex, and then QHR could form a G-quadruplex and exerted the activity. The 11.6-fold higher activity was induced by K(+) with an EC50 value of 23 mM, but not by Na(+), which is desirable when the activity switching between the intra-/extracellular environment is aimed at. This is the first report of the activation of functional RNA through a 'dual G-quadruplex formation system'.

Boosting of activity enhancement of K(+)-responsive quadruplex hammerhead ribozyme.

Two second-generation quadruplex hammerhead ribozymes, whose activity enhances in response to K(+)via quadruplex formation of embedded r(GGA)3GG, were developed. Different strategies were applied to suppress basal activity when K(+) is absent. As a result, the activity enhancement upon the addition of K(+) has reached as high as 21-fold.

An FT-IR study on packing defects in mixed ß-aggregates of poly(L-glutamic acid) and poly(D-glutamic acid): a high-pressure rescue from a kinetic trap.

Under favorable conditions of pH and temperature, poly(L-glutamic acid) (PLGA) adopts different types of secondary and quaternary structures, which include spiral assemblies of amyloid-like fibrils. Heating of acidified solutions of PLGA (or PDGA) triggers formation of ß(2)-type aggregates with morphological and tinctorial properties typical for amyloid fibrils. In contrast to regular antiparallel ß-sheet (ß(1)), the amide I' vibrational band of ß(2)-fibrils is unusually red-shifted below 1600 cm(-1), which has been attributed to bifurcated hydrogen bonds coupling C═O and N-D groups of the main chains to glutamic acid side chains. However, unlike for pure PLGA, the amide I' band of aggregates precipitating from racemic mixtures of PLGA and PDGA (ß(1)) is dominated by components at 1613 and 1685 cm(-1)-typically associated with intermolecular antiparallel ß-sheets. The coaggregation of PLGA and PDGA chains is slower and biphasic and leads to less-structured assemblies of fibrils, which is reflected in scanning electron microscopy images, sedimentation properties, and fluorescence intensity after staining with thioflavin T. The ß(1)-type aggregates are metastable, and they slowly convert to fibrils with the infrared characteristics of ß(2)-type fibrils. The process is dramatically accelerated under high pressure. This implies the presence of void volumes within structural defects in racemic aggregates, preventing the precise alignment of main and side chains necessary to zip up ladders of bifurcated hydrogen bonds. As thermodynamic costs associated with maintaining void volumes within the racemic aggregate increase under high pressure, a hyperbaric treatment of misaligned chains leads to rectifying the packing defects and formation of the more compact form of fibrils.