45 research outputs found
Non-canonical nucleosides and proto-urea-RNA at the chemical origins of life
The work presented in this dissertation was aimed towards furthering our understanding of the chemical processes that gave rise to the emergence of life on Earth. Specifically, we provide new evidence to support the RNA World Hypothesis, which suggests that the first organisms were preceded by an era in which RNA molecules catalysed their own autonomous replication. To this end, the syntheses of certain prebiotically plausible pseudo-RNA nucleosides derived from Biuret and Triuret are reported. Remarkably, in addition to being hydrolytically stable, these planar H-bonded pseudobases exhibit unique base-pairing properties that mirror that of a UG wobble-base pair when coupled with guanine or inosine within an RNA duplex. Possible mechanisms for an evolutionary transition from such pseudobases to the canonical pyrimidines are discussed. In pursuit of further proto-RNA structures, prebiotically plausible routes to a number of extant methylated nucleobases, as well as the carbamoylated adenosine nucleosides N6-threonylcarbamoyl adenosine (t6A) and N6-glycinylcarbamoyl adenosine (g6A) are described. These nucleosides are universally conserved and present within the genome of the last universal common ancestor (LUCA), which together with their ease of synthesis, suggests that they might endure today as molecular fossils from our RNA-based progenitors. Preparation of the first nucleoside phosphoramidites for g6A and related structures were also established, thus allowing their incorporation into RNA strands and investigation of their theorised role in the origin of translation. Finally, a novel prebiotically plausible route to pyrimidine nucleosides is reported, as well as encouraging preliminary results towards the synthesis of a phosphoramidite building block of the 1-(isoxazol-3-yl)-3-ribosylurea precursor from that pathway
ProtoâUreaâRNA (Wöhler RNA) Containing Unusually Stable Urea Nucleosides
The RNA world hypothesis assumes that life on Earth began with nucleotides that formed informationâcarrying RNA oligomers able to selfâreplicate. Prebiotic reactions leading to the contemporary nucleosides are now known, but their execution often requires specific starting materials and lengthy reaction sequences. It was therefore proposed that the RNA world was likely proceeded by a protoâRNA world constructed from molecules that were likely present on the early Earth in greater abundance. Herein, we show that the prebiotic starting molecules bisâurea (biuret) and trisâurea (triuret) are able to directly react with ribose. The ureaâribosides are remarkably stable because they are held together by a network of intramolecular, bifurcated hydrogen bonds. This even allowed the synthesis of phosphoramidite building blocks and incorporation of the units into RNA. Investigations of the nucleotidesâ baseâpairing potential showed that triuret:G RNA base pairs closely resemble U:G wobble base pairs. Based on the probable abundance of urea on the early Earth, we postulate that ureaâcontaining RNA bases are good candidates for a protoâRNA world
Prebiotic methylations and carbamoylations generate non-canonical RNA nucleosides as molecular fossils of an early Earth
The RNA world hypothesis assumes that life on earth started with small RNA molecules that catalyzed their own formation. Vital to this hypothesis is the need for prebiotic routes towards RNA. Contemporary RNA, however, is not only constructed from the four canonical nucleobases (A, C, G and U), but it contains in addition many chemically modified (non-canonical) bases. A yet open question is if these non-canonical bases were formed in parallel to the canonical bases (chemical origin), or whether they were created later, when life demanded higher functional diversity (biological origin). Here we show that isocyanates in combination with sodium nitrite establish methylating and carbamoylating reactivity compatible with early Earth conditions. This chemistry leads to the formation of methylated and amino acid modified nucleosides that are still extant. Our data provide a plausible scenario for the chemical origin of certain non-canonical bases, which suggests that they are fossils of an early Earth
Click Chemistry Enables Rapid Amplification of Full-Length Reverse Transcripts for Long-Read Third Generation Sequencing
Here we describe the development of a novel click chemistry-based method for the generation and amplification of full-length cDNA libraries from total RNA, while avoiding the need for problematic template-switching (TS) reactions. Compared with prior efforts, our method involves neither random priming nor stochastic cDNA termination, thus enabling amplification of transcripts that were previously inaccessible via related click chemistry-based RNA sequencing techniques. A key modification involving the use of PCR primers containing two overhanging 3âČ-nucleotides substantially improved the read-through compatibility of the 1,4-disubstituted 1,2,3-triazole-containing cDNA, where such modifications typically hinder amplification. This allowed us to more than double the possible insert size compared with the state-of-the art click chemistry-based technique, PAC-seq. Furthermore, our method performed on par with a commercially available PCR-cDNA RNA sequencing kit, as determined by Oxford Nanopore sequencing. Given the known advantages of PAC-seq, namely, suppression of PCR artifacts, we anticipate that our contribution could enable diverse applications including improved analyses of mRNA splicing variants and fusion transcripts
Unified prebiotically plausible synthesis of pyrimidine and purine RNA ribonucleotides
Theories about the origin of life require chemical pathways that allow formation of lifeâs key building blocks under prebiotically plausible conditions. Complex molecules like RNA must have originated from small molecules whose reactivity was guided by physico-chemical processes. RNA is constructed from purine and pyrimidine nucleosides, both of which are required for accurate information transfer. This is the prerequisite for Darwinian evolution. While separate pathways to purines and pyrimidines have been reported, their concurrent syntheses remain a challenge. We report the synthesis of the pyrimidine nucleosides from small molecules and ribose, driven solely by wet-dry cycles. In the presence of phosphate-containing minerals, 5â-mono- and di-phosphates also form selectively in one-pot. The pathway is compatible with purine synthesis, allowing the concurrent formation of all Watson-Crick bases
Wet-dry cycles enable the parallel origin of canonical and non-canonical nucleosides by continuous synthesis
The molecules of life were created by a continuous physicochemical process on an early Earth. In this hadean environment, chemical transformations were driven by fluctuations of the naturally given physical parameters established for example by wet-dry cycles. These conditions might have allowed for the formation of (self)-replicating RNA as the fundamental biopolymer during chemical evolution. The question of how a complex multistep chemical synthesis of RNA building blocks was possible in such an environment remains unanswered. Here we report that geothermal fields could provide the right setup for establishing wet-dry cycles that allow for the synthesis of RNA nucleosides by continuous synthesis. Our model provides both the canonical and many ubiquitous non-canonical purine nucleosides in parallel by simple changes of physical parameters such as temperature, pH and concentration. The data show that modified nucleosides were potentially formed as competitor molecules. They could in this sense be considered as molecular fossils
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Single-cell transcriptomics of suprachiasmatic nuclei reveal a Prokineticin-driven circadian network.
Circadian rhythms in mammals are governed by the hypothalamic suprachiasmatic nucleus (SCN), in which 20,000 clock cells are connected together into a powerful time-keeping network. In the absence of network-level cellular interactions, the SCN fails as a clock. The topology and specific roles of its distinct cell populations (nodes) that direct network functions are, however, not understood. To characterise its component cells and network structure, we conducted single-cell sequencing of SCN organotypic slices and identified eleven distinct neuronal sub-populations across circadian day and night. We defined neuropeptidergic signalling axes between these nodes, and built neuropeptide-specific network topologies. This revealed their temporal plasticity, being up-regulated in circadian day. Through intersectional genetics and real-time imaging, we interrogated the contribution of the Prok2-ProkR2 neuropeptidergic axis to network-wide time-keeping. We showed that Prok2-ProkR2 signalling acts as a key regulator of SCN period and rhythmicity and contributes to defining the network-level properties that underpin robust circadian co-ordination. These results highlight the diverse and distinct contributions of neuropeptide-modulated communication of temporal information across the SCN
The twilight of the Liberal Social Contract? On the Reception of Rawlsian Political Liberalism
This chapter discusses the Rawlsian project of public reason, or public justification-based 'political' liberalism, and its reception. After a brief philosophical rather than philological reconstruction of the project, the chapter revolves around a distinction between idealist and realist responses to it. Focusing on political liberalismâs critical reception illuminates an overarching question: was Rawlsâs revival of a contractualist approach to liberal legitimacy a fruitful move for liberalism and/or the social contract tradition? The last section contains a largely negative answer to that question. Nonetheless the chapter's conclusion shows that the research programme of political liberalism provided and continues to provide illuminating insights into the limitations of liberal contractualism, especially under conditions of persistent and radical diversity. The programme is, however, less receptive to challenges to do with the relative decline of the power of modern states
Effects of antiplatelet therapy on stroke risk by brain imaging features of intracerebral haemorrhage and cerebral small vessel diseases: subgroup analyses of the RESTART randomised, open-label trial
Background
Findings from the RESTART trial suggest that starting antiplatelet therapy might reduce the risk of recurrent symptomatic intracerebral haemorrhage compared with avoiding antiplatelet therapy. Brain imaging features of intracerebral haemorrhage and cerebral small vessel diseases (such as cerebral microbleeds) are associated with greater risks of recurrent intracerebral haemorrhage. We did subgroup analyses of the RESTART trial to explore whether these brain imaging features modify the effects of antiplatelet therapy