摂動を抑えた1細胞の遺伝子発現解析

京都大学新制・課程博士博士(工学)甲第23155号工博第4799号新制||工||1750(附属図書館)京都大学大学院工学研究科マイクロエンジニアリング専攻(主査)教授 横川 隆司, 教授 井上 康博, 教授 中部 主敬学位規則第4条第1項該当Doctor of Philosophy (Engineering)Kyoto UniversityDFA

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

Structural colour enhanced microfluidics

マイクロ流体デバイスの製造に革新をもたらす新手法. 京都大学プレスリリース. 2022-05-19.New process revolutionizes microfluidic fabrication. 京都大学プレスリリース. 2022-05-19.Advances in microfluidic technology towards flexibility, transparency, functionality, wearability, scale reduction or complexity enhancement are currently limited by choices in materials and assembly methods. Organized microfibrillation is a method for optically printing well-defined porosity into thin polymer films with ultrahigh resolution. Here we demonstrate this method to create self-enclosed microfluidic devices with a few simple steps, in a number of flexible and transparent formats. Structural colour, a property of organized microfibrillation, becomes an intrinsic feature of these microfluidic devices, enabling in-situ sensing capability. Since the system fluid dynamics are dependent on the internal pore size, capillary flow is shown to become characterized by structural colour, while independent of channel dimension, irrespective of whether devices are printed at the centimetre or micrometre scale. Moreover, the capability of generating and combining different internal porosities enables the OM microfluidics to be used for pore-size based applications, as demonstrated by separation of biomolecular mixtures