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

生細胞中の核酸のダイナミクスに光を当てる --生細胞中における挙動は試験管中とは異なる--. 京都大学プレスリリース. 2022-11-30.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 (kex) 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 kex 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

K⁺に応答して活性を自律的にスイッチングするインテリジェントリボザイムおよびRNAアプタマーの開発

京都大学0048新制・課程博士博士(エネルギー科学)甲第19415号エネ博第323号新制||エネ||65(附属図書館)32440京都大学大学院エネルギー科学研究科エネルギー基礎科学専攻(主査)教授 片平 正人, 教授 森井 孝, 教授 木下 正弘学位規則第4条第1項該当Doctor of Energy ScienceKyoto UniversityDGA

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

Accepted 13 Feb 2015.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

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'

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

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

An FT-IR Study on Packing Defects in Mixed β-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 β₂-type aggregates with morphological and tinctorial properties typical for amyloid fibrils. In contrast to regular antiparallel β-sheet (β₁), the amide I′ vibrational band of β₂-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 (β₁) is dominated by components at 1613 and 1685 cm^–1typically associated with intermolecular antiparallel β-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 β₁-type aggregates are metastable, and they slowly convert to fibrils with the infrared characteristics of β₂-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