Nano–bio interaction between human immunoglobulin G and nontoxic, near-infrared emitting water-borne silicon quantum dot micelles

In recent years, the field of nanomaterials has exponentially expanded with versatile biological applications. However, one of the roadblocks to their clinical translation is the critical knowledge gap about how the nanomaterials interact with the biological microenvironment (nano–bio interactions). When nanomaterials are used as drug carriers or contrast agents for biological imaging, the nano–bio interaction-mediated protein conformational changes and misfolding could lead to disease-related molecular alterations and/or cell death. Here, we studied the conformation changes of human immunoglobulin G (IgG) upon interaction with silicon quantum dots functionalized with 1-decene, Pluronic-F127 (SiQD-De/F127 micelles) using UV-visible, fluorescence steady state and excited state kinetics, circular dichroism, and molecular modeling. Decene monolayer terminated SiQDs are accumulated inside the Pluronic F127 shells to form SiQD-De/F127 micelles and were shown to bind strongly with IgG. In addition, biological evaluation studies in cell lines (HeLa, Fibroblast) and medaka fish (eggs and larvae) showed enhanced uptake and minimal cytotoxicity. Our results substantiate that engineered QDs obviating the protein conformational changes could have adept bioefficacy

An informatics approach to distinguish RNA modifications in nanopore direct RNA sequencing

Reading RNA modifications more precisely in a pocket-sized device. 京都大学プレスリリース. 2022-08-24.Modifications in RNA can influence their structure, function, and stability and play essential roles in gene expression and regulation. Methods to detect RNA modifications rely on biophysical techniques such as chromatography or mass spectrometry, which are low throughput, or on high throughput short-read sequencing techniques based on selectively reactive chemical probes. Recent studies have utilized nanopore-based fourth-generation sequencing methods to detect modifications by directly sequencing RNA in its native state. However, these approaches are based on modification-associated mismatch errors that are liable to be confounded by SNPs. Also, there is a need to generate matched knockout controls for reference, which is laborious. In this work, we introduce an internal comparison strategy termed “IndoC, ” where features such as ‘trace’ and ‘current signal intensity’ of potentially modified sites are compared to similar sequence contexts on the same RNA molecule within the sample, alleviating the need for matched knockout controls. We first show that in an IVT model, ‘trace’ is able to distinguish between artificially generated SNPs and true pseudouridine (Ψ) modifications, both of which display highly similar mismatch profiles. We then apply IndoC on yeast and human ribosomal RNA to demonstrate that previously reported Ψ sites show marked changes in their trace and signal intensity profiles compared with their unmodified counterparts in the same dataset. Finally, we perform direct RNA sequencing of RNA containing Ψ intact with a chemical probe adduct (N-cyclohexyl-N′-β-(4-methylmorpholinium) ethylcarbodiimide [CMC]) and show that CMC reactivity also induces changes in trace and signal intensity distributions in a Ψ specific manner, allowing their separation from high mismatch sites that display SNP-like behavior

Design of a new fluorescent probe: Pyrrole/imidazole hairpin polyamides with pyrene conjugation at their γ-turn.

Fluorophores that are conjugated with N-methylpyrrole-N-methylimidazole (Py-Im) polyamides postulates versatile applications in biological and physicochemical studies. Here, we show the design and synthesis of new types of pyrene-conjugated hairpin Py-Im polyamides (1-5). We evaluated the steady state fluorescence of the synthesized conjugates (1-5) in the presence and absence of oligodeoxynucleotides 5'-CGTATGGACTCGG-3' (ODN 1) and 5'-CCGAGTCCATACG-3' (ODN 2) and observed a distinct increase in emission at 386nm with conjugates 4 and 5. Notably, conjugate 5 that contains a β-alanine linker had a stronger binding affinity (K(D)=1.73×10(-8)M) than that of conjugate 4 (K(D)=1.74×10(-6)M). Our data suggests that Py-Im polyamides containing pyrene fluorophore with a β-alanine linker at the γ-turn NH(2) position can be developed as the competent fluorescent DNA-binding probes

Alteration of epigenetic program to recover memory and alleviate neurodegeneration: prospects of multi-target molecules

Accepted 12 Apr 2014.Epigenetic chromatin remodeling and signalling pathways play an integral role in transcription dependent neurodegeneration and long-term potentiation (LTP), a cellular model associated with learning and memory. Pathological epigenetic modifications associated with neurological disorders are inherently flexible and can be reversed through pharmacological intervention. Small molecules are the favored drugs for clinicians, and in neurological disorders associated with complex cellular mechanisms, epigenetic and/or signalling pathway enzymes inhibiting small molecules have shown clinical prospects. Recently, small molecules with two or more functionalities, such as sequence-specific recognition and signalling pathways and/or enzyme modulation, have shown capabilities as efficient transcriptional activators. Here, we give a balanced overview of the key factors associated with memory recovery and neurodegeneration, available chemical tools for modulation and the demand to develop next-generation small molecules with multi-functional activities to treat such intricate, multi-gene associated neurological disorders

A Multi-target Small Molecule for Targeted Transcriptional Activation of Therapeutically Significant Nervous System Genes

An integrated multi-target small molecule capable of altering dynamic epigenetic and transcription programs associated with the brain and nervous system has versatile applications in the regulation of therapeutic and cell-fate genes. Recently, we have been constructing targeted epigenetic ON switches by integrating sequence-specific DNA binding pyrrole-imidazole polyamides with a potent histone deacetylase inhibitor SAHA. Here, we identified a DNA-based epigenetic ON switch termed SAHA-L as the first-ever multi-target small molecule capable of inducing transcription programs associated with the human neural system and brain synapses networks in BJ human foreskin fibroblasts and 201B7-iPS cells. Ingenuity pathway analysis showed that SAHA-L activates the signaling of synaptic receptors like glutamate and γ-aminobutyric acid, which are key components of autism spectrum disorders. The long-term incubation of SAHA-L in 201B7-iPS cells induced morphology changes and promoted a neural progenitor state. Our finding suggests that the tunable SAHA-L could be advanced as a cell-type-independent multi-target small molecule for therapeutic and/or cell-fate gene modulation

Distinct DNA-based epigenetic switches trigger transcriptional activation of silent genes in human dermal fibroblasts.

人工スイッチを使った遺伝子コントロールに成功 -治療に役立つ可能性も- 京都大学プレスリリース. 2014-01-24.The influential role of the epigenome in orchestrating genome-wide transcriptional activation instigates the demand for the artificial genetic switches with distinct DNA sequence recognition. Recently, we developed a novel class of epigenetically active small molecules called SAHA-PIPs by conjugating selective DNA binding pyrrole-imidazole polyamides (PIPs) with the histone deacetylase inhibitor SAHA. Screening studies revealed that certain SAHA-PIPs trigger targeted transcriptional activation of pluripotency and germ cell genes in mouse and human fibroblasts, respectively. Through microarray studies and functional analysis, here we demonstrate for the first time the remarkable ability of thirty-two different SAHA-PIPs to trigger the transcriptional activation of exclusive clusters of genes and noncoding RNAs. QRT-PCR validated the microarray data, and some SAHA-PIPs activated therapeutically significant genes like KSR2. Based on the aforementioned results, we propose the potential use of SAHA-PIPs as reagents capable of targeted transcriptional activation

Chemical Probe-Based Nanopore Sequencing to Selectively Assess the RNA Modifications

Nanopore direct RNA sequencing (dRNA-Seq) reads reveal RNA modifications through consistent error profiles specific to a modified nucleobase. However, a null data set is required to identify actual RNA modification-associated errors for distinguishing it from confounding highly intrinsic sequencing errors. Here, we reveal that inosine creates a signature mismatch error in dRNA-Seq reads and obviates the need for a null data set by harnessing the selective reactivity of acrylonitrile for validating the presence of actual inosine modifications. Selective reactivity of acrylonitrile toward inosine altered multiple dRNA-Seq parameters like signal intensity and trace value. We also deduced the stoichiometry of inosine modification through deviation in signal intensity and trace value using this chemical biology approach. Furthermore, we devised Nano ICE-Seq, a protocol to overcome the low coverage issue associated with direct RNA sequencing. Taken together, our chemical probe-based approach may facilitate the knockout-free detection of disease-associated RNA modifications in clinical scenarios

Chemical Probe-Based Nanopore Sequencing to Selectively Assess the RNA Modifications

Nanopore direct RNA sequencing (dRNA-Seq) reads reveal RNA modifications through consistent error profiles specific to a modified nucleobase. However, a null data set is required to identify actual RNA modification-associated errors for distinguishing it from confounding highly intrinsic sequencing errors. Here, we reveal that inosine creates a signature mismatch error in dRNA-Seq reads and obviates the need for a null data set by harnessing the selective reactivity of acrylonitrile for validating the presence of actual inosine modifications. Selective reactivity of acrylonitrile toward inosine altered multiple dRNA-Seq parameters like signal intensity and trace value. We also deduced the stoichiometry of inosine modification through deviation in signal intensity and trace value using this chemical biology approach. Furthermore, we devised Nano ICE-Seq, a protocol to overcome the low coverage issue associated with direct RNA sequencing. Taken together, our chemical probe-based approach may facilitate the knockout-free detection of disease-associated RNA modifications in clinical scenarios

Development of biomass waste-based carbon quantum dots and their potential application as non-toxic bioimaging agents

Over recent years, carbon quantum dots (CQDs) have advanced significantly and gained substantial attention for their numerous benefits. These benefits include their simple preparation, cost-effectiveness, small size, biocompatibility, bright luminescence, and low cytotoxicity. As a result, they hold great potential for various fields, including bioimaging. A fascinating aspect of synthesizing CQDs is that it can be accomplished by using biomass waste as the precursor. Furthermore, the synthesis approach allows for control over the physicochemical characteristics. This paper unequivocally examines the production of CQDs from biomass waste and their indispensable application in bioimaging. The synthesis process involves a simple one-pot hydrothermal method that utilizes biomass waste as a carbon source, eliminating the need for expensive and toxic reagents. The resulting CQDs exhibit tunable fluorescence and excellent biocompatibility, making them suitable for bioimaging applications. The successful application of biomass-derived CQDs has been demonstrated through biological evaluation studies in various cell lines, including HeLa, Cardiomyocyte, and iPS, as well as in medaka fish eggs and larvae. Using biomass waste as a precursor for CQDs synthesis provides an environmentally friendly and sustainable alternative to traditional methods. The resulting CQDs have potential applications in various fields, including bioimaging

Integrating Epigenetic Modulators into NanoScript for Enhanced Chondrogenesis of Stem Cells

<i>N</i>-(4-Chloro-3-(trifluoro­methyl)­phenyl)-2-ethoxy­benzamide (CTB) is a small molecule that functions by altering the chromatin architecture to modulate gene expression. We report a new CTB derivative with increased solubility and demonstrate CTB’s functionality by conjugating it on the recently established NanoScript platform to enhance gene expression and induce stem cell differentiation. NanoScript is a nanoparticle-based artificial transcription factor that emulates the structure and function of transcription factor proteins (TFs) to effectively regulate endogenous gene expression. Modifying NanoScript with CTB will more closely replicate the TF structure and enhance CTB functionality and gene expression. To this end, we first conjugated CTB onto NanoScript and initiated a time-dependent increase in histone acetyl­transferase activity. Next, because CTB is known to trigger the pathway involved in regulating <i>Sox9</i>, a master regulator of chondrogenic differentiation, we modifed a <i>Sox9</i>-specific NanoScript with CTB to enhance chondrogenic gene activity and differentiation. Because NanoScript is a tunable and robust platform, it has potential for various gene-regulating applications, such as stem cell differentiation