Biology subjects

Rajamani, S.

Publications and source records attributed to Rajamani, S..

16 recordsLinked to original sources

bioRxiv2025

Characterizing mixed single chain amphiphile-based coacervates as a robust protocell system

Prebiotic soup would have been a dilute pool of various constituent chemicals that would have reacted with each other to form biologically relevant precursors during lifes origin. In this milieu, compartments formed by liquid-liquid phase separation (LLPS) are thought to have facilitated concentration of chemicals, thereby catalyzing their reactions. Towards this, various LLPS-based systems have been studied as model protocells. Relevantly, fatty acid-based (decanoic acid) coacervates have recently been explored as model protocells. As far as protocell research is concerned, fatty acids have been studied much more extensively in the context of vesicle-forming entities when compared to them resulting in coacervate systems. Furthermore, exogenous delivery and endogenous synthesis of fatty acids suggest the prevalence of single chain amphiphiles (SCAs) on the early Earth, with a greater abundance of the shorter chain length moieties. In this backdrop, we set out to fabricate robust coacervate-based protocells using SCAs that would have been readily present in a chemically heterogeneous prebiotic soup, and which could thrive under various prebiotically relevant selection pressures. Towards this, we characterized a mixed amphiphile-based coacervate system composed of nonanoic acid (NA), nonanol (NOH) and tyramine (Tyra), which could form coacervates over a broad range of pHs, temperatures, and salt concentrations. This is noteworthy as compositionally heterogenous vesicles have also been shown to have advantages over pure fatty acid vesicles. Additionally, we also demonstrate RNA sequestration in these coacervates that gets enhanced upon addition of cationic amino acids, emphasizing the importance of co-solute interactions in the prebiotic soup. Lastly, we also demonstrate nonenzymatic template-directed primer extension in these coacervates, suggesting the potential functional role of these compartments during lifes origin.

biochemistry↗

bioRxiv2025

Dynamical interactions among compositionally distinct protocell populations and its implications for evolution of early membranes

The spontaneous self-assembly of single chain amphiphiles (SCAs) would have resulted in multiple protocell species in an early-Earth niche. Considering the heterogeneity inherent in a prebiotic milieu, interactions between physicochemically distinct protocell populations was evaluated to discern if emergent properties occurred at a systems level. This study demonstrates that depending on the physicochemical properties of the membrane, interacting populations are endowed with varied emergent properties owing to their coexistence. In a multispecies paradigm involving a two-candidate protocell system, the fitter population acted as a predator and grew at the expense of the less-fit prey population. The observed growth could be attributed to the predator attaining a more robust membrane via chemical evolution. Importantly, the prey population also accrued emergent properties like molecular crowding, and coexist in balance with the predator population, without being completely outcompeted. When extrapolating these results to a three-candidate population, the outcomes were multipronged. These findings suggest a possible route for protocell membrane evolution that could have occurred even in the absence of any sophisticated protein machinery, benefitting coexisting populations, while also illustrating evolutionary trajectories that potentially resulted in functionally complex protocells.

biophysics↗

bioRxiv2024

Cross-catalytic enhancement of peptides and RNA from a common prebiotic activated intermediate

Previous origin of life studies have demonstrated the presence of amino acids and nucleotides in the same prebiotic milieu. In this study, we set out to understand the interplay of amino acids with linear or cyclic nucleotides under prebiotically pertinent reaction conditions, especially for its implications for biomolecular evolution. We characterized the cross-catalytic effect of oligomerization, potentially stemming from the simultaneous presence of these two biochemically important monomers. Qualitative and quantitative analysis indicated the formation of longer AMP oligomers and peptides, with 8-10 fold increase in specific reaction scenarios, when compared to reactions that evaluated the monomer oligomerization in isolation. The reason behind such an increase in yield and length, in case of both the oligomers, was the formation of a reactive intermediate. This aminoacylated-AMP (AMP-aa) resulted from a condensation reaction between the nucleotide and the amino acid. We extended this to other amino acids with different R chain characteristics, to comprehend the properties required for the formation of AMP-aa under our reaction conditions. The nonenzymatic formation of these aminoacylated AMP, which in turn resulted in longer oligomers, indicates the plausibility of the emergence of initial steps involved in a primordial translation system.

biochemistry↗

bioRxiv2023

Effect of 'spent'nucleotides' on nonenzymatic RNA replication

Nonenzymatic template-directed replication would have been affected by co-solutes in a heterogenous prebiotic soup due to lack of enzymatic machinery. Unlike in contemporary biology, these reactions use chemically-activated nucleotides, which undergo rapid hydrolysis forming nucleoside monophosphates ( spent monomers). These co-solutes cannot extend the primer but continue to base pair with the template, thereby interfering with replication. We therefore aimed to understand how a mixture of spent ribonucleotides would affect nonenzymatic replication. We observed inhibition of replication in presence of the mixture, wherein predominant contribution came from the cognate Watson-Crick monomer, showing potential sequence dependence. Our study highlights how nonenzymatic RNA replication would have been directly affected by co-solutes, with ramifications for the emergence of functional polymers in an RNA World.

biochemistry↗

bioRxiv2023

Nucleotide-protocell interactions: A reciprocal relationship in prebiotically pertinent environments

Spontaneous interactions between nucleotides and lipid membranes are likely to have played a prominent role in the emergence of life on Earth. However, the effect of nucleotides on the physicochemical properties of model protocellular membranes is relatively less understood. To this end, we aimed to discern the effect of canonical nucleotides on the properties of single-chain amphiphile membranes under prebiotically relevant conditions of multiple wet-dry cycles. Furthermore, the change in critical aggregation concentration of the membranes, and their stability in the presence of nucleotides, was also investigated in Astrobiologically analogue environments. We report that different nucleotides, lipid headgroups, and the ionic makeup of the system affect lipid-nucleotide interactions, which in turn can modulate the effect of nucleotides on the membranes. Specifically, oleic acid membranes seemingly showed an increase in the critical aggregation concentration, and an increase in the stability against prebiotically relevant selection pressures, in the presence of certain nucleotides. Our work has implications for understanding how nucleotides might have shaped the protocellular landscape of the prebiotic Earth.

biochemistry↗

bioRxiv2023

A prebiotic genetic alphabet as an early Darwinian ancestor for pre-RNA evolution

RNA-based genetic code is thought to be central to lifes emergence due to its dual ability for information transfer and catalysis. Nonetheless, the genetic code of early life was potentially not restricted to canonical genetic alphabets alone. The presence of an extensive repertoire of modified nucleobases in extant biology as signatures of the past, highlights the relevance of non-canonical alphabets, ably strengthened by experiments demonstrating their ready conversion into nucleosides and nucleotides. All these strongly support a pre-RNA World, wherein informational molecules are posited to have contained alternate genetic alphabets. Nevertheless, understanding pre-RNA molecules capacity to acquire emergent function has remained less prevalent. Further, the steps involved in their transition to a canonical RNA World has not been systematically studied in the origins of life framework. In this study, we report the synthesis of a prebiotically relevant genetic alphabet containing the non-canonical nucleobase, barbituric acid. We demonstrate for the first instance the enzymatic incorporation of this prebiotically plausible alphabet (BaTP) into an RNA, using proteinaceous T7 RNA polymerase. Pertinently, the incorporation of this genetic alphabet into a baby spinach aptamer did not affect its overall secondary structure, while also allowing it to retain its aptameric function. Furthermore, we demonstrate the faithful transfer of genetic information from pre-RNA-containing barbitudine nucleotides to DNA, using a high-fidelity RNA-dependent DNA polymerase. These findings allude to a putative pathway for the early molecular evolution of the genetic code of extant life.

biochemistry↗

bioRxiv2022

TREK-1 knock-down in human atrial fibroblasts leads to a myofibroblastic phenotype: a role in phenoconversion and overview of mechano-sensitive channel mRNA expression in cardiac diseases.

Cardiac cell mechanical environment changes on a beat-by-beat basis and also in the course of various cardiac diseases. Cells sense and adapt to such mechanical cues via specialized mechano-sensors mediating adaptive signaling cascades. Here, we report TREK-1 mRNA expression and activity in human atrial fibroblasts and reveal a cross talk between TREK-1 and fibroblast phenoconversion. With the aim to reveal additional candidates underlying mechano-transduction relevant to cardiac diseases, we investigated mechano-sensitive ion channels (MSC) mRNA expression in diseased and non-diseased human hearts. Our results showed higher TREK-1 expression and activity in fibroblasts compared to myofibroblasts and we found that TREK-1 down-regulation leads a more myofibroblastic phenotype suggesting a role for this mechano-sensor in phenoconversion. In addition, TREK-1 is preferentially expressed in the left atrium compared to the right one and its expression is not significantly changed when fibroblasts from patients in sinus rhythm vs. sustained atrial fibrillation are compared. At the whole-heart level, numerous MSC were differentially expressed between atrial and ventricular or between non-diseased and diseased tissue samples. Thus, we identify atrial fibroblast-specific TREK-1 expression and activity and reveal a role of TREK-1 in atrial fibroblast pheno-conversion. We also provide a comprehensive overview of cardiac MSC mRNA expression in atrial and ventricular tissue from diseased and non-diseased patients, identifying potential novel candidates underlying mechanotransduction in cardiac diseases.

cell biology↗

bioRxiv2022

Nonenzymatic template-directed replication using 2'-3' cyclic nucleotides under wet-dry cycles

RNA World Hypothesis is centred around the idea of a period in the early history of lifes origin, wherein nonenzymatic oligomerization and replication of RNA resulted in functional ribozymes. Previous studies in this endeavour have demonstrated template-directed replication using chemically modified nucleotides and primers. Nonetheless, similar studies that used non-activated nucleotides led to the formation of RNA only with abasic sites. In this study, we report template-directed replication with prebiotically relevant cyclic nucleotides, under dehydration-rehydration (DH-RH) cycles occurring at high temperature (90{degrees}C) and alkaline conditions (pH 8). 2'-3' cyclic nucleoside monophosphates (cNMP) resulted in primer extension, while 3'-5' cNMP failed to do so. Intact extension of up to two nucleotide additions was observed with both canonical hydroxy-terminated (OH-primer) and activated amino-terminated (NH2-primer) primers. We demonstrate primer extension reactions using both purine and pyrimidine 2'-3' cNMPs, with higher product yield observed during cAMP additions. Further, the presence of lipid was observed to significantly enhance the extended product in cCMP reactions. In all, our study provides a proof-of-concept for nonenzymatic replication of RNA, using intrinsically activated prebiotically relevant cyclic nucleotides as monomers.

biochemistry↗

bioRxiv2022

pH-responsive self-assembled compartments as tuneable model protocellular membrane systems

Prebiotically plausible single-chain amphiphiles are enticing as model protocellular compartments to study the emergence of cellular life owing to their self-assembling properties. Here, we investigated the self-assembly behaviour of mono-N-dodecyl phosphate (DDP) and mixed systems of DDP with dodecyl 1-dodecanol (DOH) at varying pH conditions. Membranes composed of DDP showed pH-responsive vesicle formation in a wide range of pH with a low critical bilayer concentration (CBC). Further, the addition of DOH to DDP membrane system enhanced vesicle formation and stability in alkaline pH regimes. We also compared the high-temperature behaviour of DDP and DDP-DOH membranes with conventional fatty acid membranes. Both, DDP and DDP:DOH mixed membranes possess packing that is similar to decanoic acid membrane. However, the micropolarity of these systems are similar to phospholipid membranes. Finally, the pH-dependent modulation of different phospholipid membranes doped with DDP was also demonstrated to engineer tuneable membranes with potential translational implications.

biochemistry↗

bioRxiv2021

Porphyrin in prebiotic catalysis: Ascertaining a route for the emergence of early metalloporphyrins

Metal ions are known to catalyze certain prebiotic reactions. However, the transition from metal ions to extant metalloenzymes remains unclear. Porphyrins are found ubiquitously in the catalytic core of many ancient metalloenzymes. In this study, we evaluated the influence of porphyrin-based organic scaffold, on the catalysis, emergence and putative molecular evolution of prebiotic metalloporphyrins. We studied the effect of porphyrins on the transition metal ion-mediated oxidation of hydroquinone (HQ). We report a change in the catalytic activity of the metal ions in the presence of porphyrin. This was observed to be facilitated by the coordination between metal ions and porphyrins or by formation of non-coordinated complexes. The metal-porphyrin complexes also oxidized NADH, underscoring its versatility at oxidizing more than one substrate. Our study highlights the selective advantage that some of the metal ions would have had in the presence of porphyrin, underscoring their role in shaping the evolution of protometalloenzymes.

biochemistry↗

bioRxiv2020

Functional modeling of NMIHBA-causing PRUNE1 variants reveals a requirement for its exopolyphosphatase activity.

Neurodevelopmental disorder with microcephaly, hypotonia, and variable brain anomalies (NMIHBA) is an autosomal recessive neurodevelopmental and neurodegenerative disorder characterized by global developmental delay and severe intellectual disability. Microcephaly, progressive cortical atrophy, cerebellar hypoplasia and delayed myelination are neurological hallmarks in affected individuals. NMIHBA is caused by biallelic variants in PRUNE1 encoding prune exopolyphosphatase 1. We provide in-depth clinical description of two affected siblings harboring compound heterozygous variant alleles, c.383G>A (p.Arg128Gln), c.520G>T (p.Gly174*) in PRUNE1. To gain insights into disease biology, we biochemically characterized missense variants within the conserved N-terminal aspartic acid-histidine-histidine (DHH) motif and provide evidence that they result in the destabilization of protein structure and/or loss of exopolyphosphatase activity. Genetic ablation of Prune1 results in midgestational lethality in mice, associated with perturbations to embryonic growth and vascular development. Our findings suggest that NMIHBA results from hypomorphic variant alleles in humans and underscore the potential key role of PRUNE1 exopolyphoshatase activity in neurodevelopment.

genetics↗

bioRxiv2020

Emergence of ribozyme and tRNA-like structures from mineral-rich muddy pools on prebiotic earth

AO_SCPLOWBSTRACTC_SCPLOWThe RNA world hypothesis, although a viable one regarding the origin of life on earth, has so far failed to provide a compelling explanation for the synthesis of RNA molecules with catalytic functions, from free nucleotides via abiotic processes. To tackle this long-standing problem, we develop a realistic model for the onset of the RNA world, using experimentally determined rates for polymerization reactions. We start with minimal assumptions about the initial state that only requires the presence of short oligomers or just free nucleotides and consider the effects of environmental cycling by dividing a day into a dry, semi-wet and wet phases that are distinguished by the nature of reactions they support. Long polymers, with maximum lengths sometimes exceeding 100 nucleotides, spontaneously emerge due to a combination of non-enzymatic, non-templated polymer extension and template-directed primer extension processes. The former helps in increasing the lengths of RNA strands, whereas the later helps in producing complementary copies of the strands. Strands also undergo hydrolysis in a structure-dependent manner that favour breaking of bonds connecting unpaired nucleotides. We identify the most favourable conditions needed for the emergence of ribozyme and tRNA-like structures and double stranded RNA molecules, classify all RNA strands on the basis of their secondary structures and determine their abundance in the population. Our results indicate that under suitable environmental conditions, non-enzymatic processes would have been sufficient to lead to the emergence of a variety of ribozyme-like molecules with complex secondary structures and potential catalytic functions.

biophysics↗

bioRxiv2019

Geochemical influences on nonenzymatic oligomerization of prebiotically relevant cyclic nucleotides

The spontaneous emergence of RNA on the early Earth continues to remain an enigma in the field of origins of life. Few studies have looked at the nonenzymatic oligomerization of cyclic nucleotides under neutral to alkaline conditions, in fully dehydrated state. Herein, we systematically investigated the oligomerization of cyclic nucleotides under prebiotically relevant conditions, where starting reactants were subjected to repeated dehydration-rehydration (DH-RH) regimes, like they would have been on an early Earth. DH-RH conditions, a recurring geological theme, are driven by naturally occurring processes including diurnal cycles and tidal pool activity. These conditions have been shown to facilitate uphill oligomerization reactions in terrestrial geothermal niches, which are hypothesized to be pertinent sites for the emergence of life. 2'-3' and 3'-5' cyclic nucleotides of one purine-based (adenosine) and one pyrimidine-based (cytidine) system were evaluated in this study. Additionally, the effect of amphiphiles was also investigated. Furthermore, to discern the effect of realistic conditions on this process, the reactions were also performed using hot spring water samples from an early Earth analogue environment. Our results showed that the oligomerization of cyclic nucleotides under DH-RH conditions resulted in intact informational oligomers. Amphiphiles increased the stability of, both, the starting monomers and the resultant oligomers. In analogue condition reactions, oligomerization of nucleotides and back-hydrolysis of the resultant oligomers was pronounced. Altogether, this study demonstrates how nonenzymatic oligomerization of cyclic purine and pyrimidine nucleotides, under laboratory-simulated and early Earth analogous conditions, could have resulted in RNA oligomers of a putative RNA World.

biochemistry↗

bioRxiv2019

Compositional heterogeneity of protocellular membranes: Implications for emergence of mixed vesicular systems

Protocells are primitive cellular entities that are thought to have emerged during the dawn of life on Earth. Their membranes are considered to be made up of mixtures of single chain amphiphiles, such as fatty acids and their derivatives, moieties that would have been part of the complex prebiotic chemical landscape. In addition to their composition, the physico-chemical properties of these prebiological membranes would have been significantly affected and regulated by the physical environment that they were present in. In this study, the physico-chemical properties of two different chain length membrane systems were systematically characterized, under pertinent early Earth conditions. The membrane systems were designed to comprise a fatty acid and its alcohol and/or glycerol monoester derivative, to make a range of binary and tertiary vesicle combinations. Their properties were then evaluated as a function of multiple factors including their composition, stability under varying pH, Mg2+ ion concentrations and dilution regimes, and their permeability to small molecules. Our results demonstrate how these environmental constraints would have acted as important prebiotic selection pressures to shape the evolution of prebiological membranes. This study also illustrates how different fatty acid derivatives confer varying degree of stability when combined with their respective fatty acid moiety. Interestingly, when the membrane systems were subjected to multiple selection pressures in a consecutive manner, only the heterogeneous membrane systems survived the race. Our results illustrate that compositionally diverse membrane systems are more stable and robust to multiple selection pressures, thereby making them more suitable for supporting protocellular life.\n\nSignificance statementThe physico-chemical environment would have played an important role in shaping the composition and evolution of primitive membrane systems. This study demonstrates the importance of compositional heterogeneity on the stability of protomembrane systems under pertinent prebiotic selection pressures. Two different fatty acid based systems were mixed with their respective alcohol and/or glycerol monoester derivatives, to generate combinations of binary and tertiary membrane systems. Increasing chain length effected the physical property of the membranes thus affecting their stability. Furthermore, we also demonstrate how the head groups of the derivatives employed in this study (e.g. glycerol monoester and alcohol) contribute differently towards stabilizing the mixed membranes under a given selection condition, by discerning the molecular mechanism underlying this process. Our results illustrate how multiple selection pressures would have preferentially supported the emergence of compositionally heterogeneous membrane systems.

biochemistry↗

bioRxiv2019

NMR-based analysis of nucleotide π-stacking in a crowded environment: Implications for prebiotic reactions

The inherent heterogeneity of the prebiotic milieu is often overlooked when studying nonenzymatic reactions. However, it is important to note that the prebiotic soup of a putative RNA World would have been replete with a plethora of molecules resulting from complex chemical syntheses, as well as exogeneous delivery. The presence of such background molecules could lead to pertinent phenomenon such as molecular crowding, which can potentially affect how a reaction would advent in a crowded milieu. In the current study, we have analyzed the effect of crowding on the stacking ability of the RNA monomers, using Nuclear Magnetic Resonance (NMR) spectroscopy. Our findings corroborate that the purine monomers possess better stacking efficiency than pyrimidine based monomers. Significantly, this competence is further enhanced in the presence of a crowding agent. Interestingly, this enhanced stacking could result in higher sequestration of the purine monomers, putting their ready availability for relevant nonenzymatic polymerization and replication reactions into question. Taken together, this study demonstrates the need for systematic biophysical characterization of molecular crowding in the context of prebiotically pertinent processes. Unravelling such phenomena is essential to gather a real understanding of how the transition from abiotic to biotic, would have happened during the origin of life.

biophysics↗

bioRxiv2017

Mutations in RPL3L and MYZAP increase risk of atrial fibrillation

We performed a meta-analysis of genome-wide association studies on atrial fibrillation (AF) among 14,710 cases and 373,897 controls from Iceland and 14,792 cases and 393,863 controls from the UK Biobank, focusing on low frequency coding and splice mutations, with follow-up in samples from Norway and the US. We observed associations with two missense (OR=1.19 for both) and one splice-donor mutation (OR=1.52) in RPL3L, encoding a ribosomal protein primarily expressed in skeletal muscle and heart. Analysis of 167 RNA samples from the right atrium revealed that the splice donor mutation in RPL3L results in exon skipping. AF is the first disease associated with RPL3L and RPL3L is the first ribosomal gene implicated in AF. This finding is consistent with tissue specialization of ribosomal function. We also found an association with a missense variant in MYZAP (OR=1.37), encoding a component of the intercalated discs of cardiomyocytes, the organelle harbouring most of the mutated proteins involved in arrhythmogenic right ventricular cardiomyopathy. Both discoveries emphasize the close relationship between the mechanical and electrical function of the heart.

genetics↗