Prebiological Membranes and Their Role in the Emergence of Early Cellular Life.

Membrane compartmentalization is a fundamental feature of contemporary cellular life. Given this, it is rational to assume that at some stage in the early origins of life, membrane compartments would have potentially emerged to form a dynamic semipermeable barrier in primitive cells (protocells), protecting them from their surrounding environment. It is thought that such prebiological membranes would likely have played a crucial role in the emergence and evolution of life on the early Earth. Extant biological membranes are highly organized and complex, which is a consequence of a protracted evolutionary history. On the other hand, prebiotic membrane assemblies, which are thought to have preceded sophisticated contemporary membranes, are hypothesized to have been relatively simple and composed of single chain amphiphiles. Recent studies indicate that the evolution of prebiotic membranes potentially resulted from interactions between the membrane and its physicochemical environment. These studies have also speculated on the origin, composition, function and influence of environmental conditions on protocellular membranes as the niche parameters would have directly influenced their composition and biophysical properties. Nonetheless, the evolutionary pathways involved in the transition from prebiological membranes to contemporary membranes are largely unknown. This review critically evaluates existing research on prebiotic membranes in terms of their probable origin, composition, energetics, function and evolution. Notably, we outline new approaches that can further our understanding about how prebiotic membranes might have evolved in response to relevant physicochemical parameters that would have acted as pertinent selection pressures on the early Earth.

A versatile toolbox for posttranscriptional chemical labeling and imaging of RNA.

Cellular RNA labeling strategies based on bioorthogonal chemical reactions are much less developed in comparison to glycan, protein and DNA due to its inherent instability and lack of effective methods to introduce bioorthogonal reactive functionalities (e.g. azide) into RNA. Here we report the development of a simple and modular posttranscriptional chemical labeling and imaging technique for RNA by using a novel toolbox comprised of azide-modified UTP analogs. These analogs facilitate the enzymatic incorporation of azide groups into RNA, which can be posttranscriptionally labeled with a variety of probes by click and Staudinger reactions. Importantly, we show for the first time the specific incorporation of azide groups into cellular RNA by endogenous RNA polymerases, which enabled the imaging of newly transcribing RNA in fixed and in live cells by click reactions. This labeling method is practical and provides a new platform to study RNA in vitro and in cells.

A clickable UTP analog for the posttranscriptional chemical labeling and imaging of RNA.

The development of robust tools and practical RNA labeling strategies that would facilitate the biophysical analysis of RNA in both cell-free and cellular systems will have profound implications in the discovery of new RNA diagnostic tools and therapeutic strategies. In this context, we describe the development of a new alkyne-modified UTP analog, 5-(1,7-octadinyl)uridine triphosphate (ODUTP), which serves as an efficient substrate for the introduction of a clickable alkyne label into RNA transcripts by bacteriophage T7 RNA polymerase and mammalian cellular RNA polymerases. The ODU-labeled RNA is effectively used by reverse transcriptase to produce cDNA, a property which could be utilized in expanding the chemical space of a RNA library in the aptamer selection scheme. Further, the alkyne label on RNA provides a convenient tool for the posttranscriptional chemical functionalization with a variety of biophysical tags (fluorescent, affinity, amino acid and sugar) by using alkyne-azide cycloaddition reaction. Importantly, the ability of endogenous RNA polymerases to specifically incorporate ODUTP into cellular RNA transcripts enabled the visualization of newly transcribing RNA in cells by microscopy using click reactions. In addition to a clickable alkyne group, ODU contains a Raman scattering label (internal disubstituted alkyne), which exhibits characteristic Raman shifts that fall in the Raman-silent region of cells. Our results indicate that an ODU label could potentially facilitate two-channel visualization of RNA in cells by using click chemistry and Raman spectroscopy. Taken together, ODU represents a multipurpose ribonucleoside tool, which is expected to provide new avenues to study RNA in cell-free and cellular systems.

A Prebiotic Genetic Nucleotide as an Early Darwinian Ancestor for Pre-RNA Evolution.

Prebiotic genetic nucleotides (PGNs) often outcompete canonical alphabets in the formation of nucleotides and subsequent RNA oligomerization under early Earth conditions. This indicates that the early genetic code might have been dominated by pre-RNA that contained PGNs for information transfer and catalysis. Despite this, deciphering pre-RNAs' capacity to acquire function and delineating their evolutionary transition to a canonical RNA World has remained under-researched in the origins of life (OoL) field. We report the synthesis of a prebiotically relevant nucleotide (BaTP) containing the noncanonical nucleobase barbituric acid. We demonstrate the first instance of its enzymatic incorporation into an RNA, using a T7 RNA polymerase. BaTP's incorporation into baby spinach aptamer allowed it to retain its overall secondary structure and function. Finally, we also demonstrate faithful transfer of information from the pre-RNA-containing BaTP to DNA, using a high-fidelity RNA-dependent DNA polymerase, alluding to how selection pressures and complexities could have ensued during the molecular evolution of the early genetic code.

Spontaneous emergence of membrane-forming protoamphiphiles from a lipid-amino acid mixture under wet-dry cycles.

Dynamic interplay between peptide synthesis and membrane assembly would have been crucial for the emergence of protocells on the prebiotic Earth. However, the effect of membrane-forming amphiphiles on peptide synthesis, under prebiotically plausible conditions, remains relatively unexplored. Here we discern the effect of a phospholipid on peptide synthesis using a non-activated amino acid, under wet-dry cycles. We report two competing processes simultaneously forming peptides and N-acyl amino acids (NAAs) in a single-pot reaction from a common set of reactants. NAA synthesis occurs via an ester-amide exchange, which is the first demonstration of this phenomenon in a lipid-amino acid system. Furthermore, NAAs self-assemble into vesicles at acidic pH, signifying their ability to form protocellular membranes under acidic geothermal conditions. Our work highlights the importance of exploring the co-evolutionary interactions between membrane assembly and peptide synthesis, having implications for the emergence of hitherto uncharacterized compounds of unknown prebiotic relevance.

Imaging Newly Transcribed RNA in Cells by Using a Clickable Azide-Modified UTP Analog.

Robust RNA labeling and imaging methods that enable the understanding of cellular RNA biogenesis and function are highly desired. In this context, we describe a practical chemical labeling method based on a bioorthogonal reaction, namely, azide-alkyne cycloaddition reaction, which facilitates the fluorescence imaging of newly transcribed RNA in both fixed and live cells. This strategy involves the transfection of an azide-modified UTP analog (AMUTP) into mammalian cells, which gets specifically incorporated into RNA transcripts by RNA polymerases present inside the cells. Subsequent posttranscriptional click reaction between azide-labeled RNA transcripts and a fluorescent alkyne substrate enables the imaging of newly synthesized RNA in cells by confocal microscopy. Typically, 50 µM to 1 mM of AMUTP and a transfection time of 15-60 min produce significant fluorescence signal from labeled RNA transcripts in cells.

Enzymatic incorporation of an azide-modified UTP analog into oligoribonucleotides for post-transcriptional chemical functionalization.

This protocol describes the detailed experimental procedure for the synthesis of an azide-modified uridine triphosphate analog and its effective incorporation into an oligoribonucleotide by in vitro transcription reactions. Furthermore, procedures for labeling azide-modified oligoribonucleotides post-transcriptionally with biophysical probes by copper(I)-catalyzed alkyne-azide cycloaddition (CuAAC) and Staudinger reactions are also provided. This post-transcriptional chemical modification protocol is simple and modular, and it affords labeled oligonucleotides in reasonable amounts for biophysical assays. The procedure for enzymatic incorporation of the monophosphate of azide-modified UTP into an oligoribonucleotide transcript takes ∼2 d, and subsequent post-transcriptional chemical functionalization of the transcript takes about 2 d.

Posttranscriptional chemical functionalization of azide-modified oligoribonucleotides by bioorthogonal click and Staudinger reactions.

Direct incorporation of azide groups into RNA oligonucleotides by in vitro transcription reactions in the presence of a new azide-modified UTP analogue, and subsequent posttranscriptional chemical labeling of azide-modified oligoribonucleotide transcripts by click and Staudinger reactions are described. This postsynthetic labeling protocol is robust and modular, and offers an alternative access to RNA labeled with biophysical probes.