Biology subjects

Devaraj, N. K.

Publications and source records attributed to Devaraj, N. K..

17 recordsLinked to original sources

bioRxiv2026

N-Terminal Alkylation of Proteins with Triazole-4-carbaldehyde for Targeted Liposome Engineering

A platform method to associate native proteins and peptides onto a liposome membrane using site-specific N-terminal alkylation based on 1H-1,2,3-triazole-4-carbaldehyde (TA4C) is developed. The TA4C reagent reacts with the N-terminal -amino group of native proteins under mild aqueous conditions in a single step, without genetic engineering or protecting group strategies. Equipping TA4C with hexyl and nonyl chains provides a direct handle for tuning the association between the protein and membrane. N-terminal alkylation of green fluorescent protein (GFP) as a model proceeds in high yield (93% for the hexyl group and 70% for the nonyl group), and tethering the N-terminal alkyl group on GFP efficiently associates the protein with the liposomal membrane, as confirmed by confocal laser scanning microscopy and dynamic light scattering. We extended this strategy to an investigation of the GE11 peptide, a ligand for the epidermal growth factor receptor (EGFR). The liposome immobilized with GE11 peptide possessing an N-terminal alkyl group enables active targeting with EGFR-overexpressing A431 cells. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=80 SRC="FIGDIR/small/742023v1_ufig1.gif" ALT="Figure 1"> View larger version (20K): org.highwire.dtl.DTLVardef@9c5ec3org.highwire.dtl.DTLVardef@117765org.highwire.dtl.DTLVardef@b5c471org.highwire.dtl.DTLVardef@1741293_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2025

1-Deoxysphingolipids dysregulate membrane properties and cargo trafficking in the early secretory pathway

1-Deoxysphingolipids are non-canonical sphingolipids linked to several diseases, but their cellular effects are poorly understood. Here, we utilize lipid chemical biology approaches to investigate the role of 1-deoxysphingolipid metabolism on the properties and functions of secretory membranes. We first applied organelle-specific bioorthogonal labeling to visualize the subcellular distribution of metabolically tagged 1-deoxysphingolipids in RPE-1 cells, observing that they are retained in the endoplasmic reticulum (ER). We found that 1-deoxysphingolipids can be transported by the non-vesicular transporter CERT in vitro but are retained at ER exit sites (ERES) in cells, suggesting that they do not efficiently sort into vesicular carriers. Cells expressing disease-associated variants of serine palmitoyl-CoA transferase (SPT) accumulated long-chain 1-deoxysphingolipids, which reduced ER membrane fluidity and enlarged ERES. We observed that the rates of membrane protein release from the ER were altered in response to mutant SPT expression, in a manner that was dependent on the cargo affinity for ordered or disordered membranes. We propose that dysregulation of sphingolipid metabolism alters secretory membrane properties, which can then modulate protein trafficking.

cell biology↗

bioRxiv2024

Subcellular imaging of lipids and sugars using genetically encoded proximity sensors

The lipid composition of cells varies widely across organelles and between individual membrane leaflets. Transport proteins are thought to generate this heterogeneity, but measuring their functions in vivo has been hampered by limited tools for imaging lipids at relevant spatial resolutions. Here we present fluorogen-activating coincidence encounter sensing (FACES), a chemogenetic tool capable of quantitatively imaging subcellular lipid pools and reporting their transbilayer orientation in living cells. FACES combines bioorthogonal chemistry with genetically encoded fluorogen-activating proteins (FAPs) for reversible proximity sensing of conjugated molecules. We first apply this approach to identify roles for lipid transfer proteins that traffic phosphatidylcholine pools between the ER and mitochondria. We then show that transmembrane domain-containing FAPs can reveal the membrane asymmetry of multiple lipid classes in the trans-Golgi network and be used to investigate the mechanisms that generate it. Lastly, we demonstrate FACES can be applied to measure other molecule classes, like sugars.

cell biology↗

bioRxiv2023

Site-specific Covalent Labeling of DNA Substrates by an RNA Transglycosylase

Bacterial tRNA guanine transglycosylases (TGTs) catalyze the exchange of guanine for the 7-deazaguanine queuine precursor, prequeuosine1 (preQ1). While the native nucleic acid substrate for bacterial TGTs is the anticodon loop of queuine-cognate tRNAs, the minimum recognition sequence for the enzyme is a structured hairpin containing the target G nucleobase in a "UGU" loop motif. Previous work has established an RNA modification system, RNA-TAG, in which E. coli TGT exchanges the target G on an RNA of interest for chemically modified preQ1 substrates linked to a small molecule reporter such as biotin or a fluorophore. While extending the substrate scope of RNA transglycosylases to include DNA would enable numerous applications, it has been previously reported that TGT is incapable of modifying native DNA. Here we demonstrate that TGT can in fact recognize and label specific DNA substrates. Through iterative testing of rationally mutated DNA hairpin sequences, we determined the minimal sequence requirements for transglycosylation of unmodified DNA by E. coli TGT. Controlling steric constraint in the DNA hairpin dramatically affects labeling efficiency, and, when optimized, can lead to near quantitative site-specific modification. We demonstrate the utility of our newly developed DNA-TAG system by rapidly synthesizing probes for fluorescent Northern blotting of spliceosomal U6 RNA and RNA FISH visualization of the long noncoding RNA, MALAT1. The ease and convenience of the DNA-TAG system will provide researchers with a tool for accessing a wide variety of affordable modified DNA substrates.

synthetic biology↗

bioRxiv2021

Functionalizing lipid sponge droplets with DNA

Nucleic acids are among the most versatile molecules for the construction of biomimetic systems because they can serve as information carriers and programmable construction materials. How nucleic acids interact with membranous coacervate compartments such as lipid sponge droplets is not known. Here we systematically characterize the potential of DNA to functionalize lipid sponge droplets and demonstrate a strong size dependence for sequestration into the sponge phase. Double stranded DNA molecules of more than 300 bp are excluded and form a corona on the surface of droplets they are targeted to. Shorter DNA molecules partition efficiently into the lipid sponge phase and can direct DNA-templated reactions to droplets. We demonstrate repeated capture and release of labeled DNA strands by dynamic hybridization and strand displacement reactions that occur inside droplets. Our system opens new opportunities for DNA-encoded functions in lipid sponge droplets such as cargo control and signaling.

synthetic biology↗

bioRxiv2021

Semisynthesis of functional transmembrane proteins in GUVs

Cellular transmembrane (TM) proteins are essential sentries of the cell facilitating cell-cell communication, internal signaling, and solute transport. Reconstituting functional TM proteins into model membranes remains a challenge due to the difficulty of expressing hydrophobic TM domains and the required use of detergents. Herein, we use a intein-mediated ligation strategy to semisynthesize bitopic TM proteins in synthetic membranes. We have adapted the trans splicing capabilities of split inteins for a native peptide ligation between a synthetic TM peptide embedded in the membrane of giant unilamellar vesicles (GUVs) and an expressed soluble protein. We demonstrate that the extracellular domain of programmed cell death protein 1 (PD-1), a mammalian transmembrane immune checkpoint receptor, retains its function for binding its ligand PD-L1 at a reconstituted membrane interface after ligation to a synthetic TM peptide in GUV membranes. We envision that the construction of full-length TM proteins using orthogonal split intein-mediated semisynthetic protein ligations will expand applications of membrane protein reconstitution in pharmacology, biochemistry, biophysics, and artificial cell development.

biochemistry↗

bioRxiv2021

RNA-CLAMP Enables Photo-activated Control of CRISPR-Cas9 Gene Editing by Site-specific Intramolecular Cross-linking of the sgRNA

Here we introduce RNA-CLAMP, a technology which enables site-specific and enzymatic cross-linking (clamping) of two selected stem loops within an RNA of interest. Intramolecular clamping of the RNA can disrupt normal RNA function, whereas subsequent photo-cleavage of the crosslinker restores activity. We applied the RNA-CLAMP technique to the single guide RNA of the CRISPR-Cas9 gene editing system. By clamping two stem loops of the single-guide RNA (sgRNA) with a photo-cleavable cross-linker, gene editing was completely silenced. Visible light irradiation cleaved the crosslinker and restored gene editing with high spatiotemporal resolution. Furthermore, by designing two photo-cleavable linkers which are responsive to different wavelength of lights, we achieved multiplexed photo-activation of gene editing in mammalian cells. Notably, although the Cas9-sgRNA RNP is not capable of DNA cleavage activity upon clamping, it maintained the capability to bind to the target DNA. The RNA-CLAMP enabled photo-activated CRISPR-Cas9 gene editing platform offers clean background, free choice of activation wavelength and multiplexing capability. We believe that this technology to precisely and rapidly control gene editing will serve as a versatile tool in the future development of stimuli responsive gene editing technologies. Beyond gene editing, RNA-CLAMP provides a site-specific tool for manipulating the internal structure of functional RNAs.

biochemistry↗

bioRxiv2021

Expression of fatty acyl-CoA ligase drives one-pot de novo synthesis of membrane-bound vesicles in a cell free transcription-translation system

Despite the central importance of lipid membranes in cellular organization, it is challenging to reconstitute their de novo formation from minimal chemical and biological elements. Here we describe a chemoenzymatic route to membrane-forming non-canonical phospholipids in which cysteine-modified lysolipids undergo spontaneous coupling with fatty acyl-CoA thioesters generated enzymatically by a fatty acyl-CoA ligase. Due to the high efficiency of the reaction, we were able to optimize phospholipid membrane formation in a cell-free transcription-translation (TX-TL) system. Combining DNA encoding for the fatty acyl-CoA ligase with suitable lipid precursors, enabled spontaneous one-pot de novo synthesis of membrane-bound vesicles. Non-canonical sphingolipid synthesis was also possible by using a cysteine-modified lysosphingomyelin as a precursor. When the sphingomyelin-interacting protein lysenin is co-expressed alongside the acyl CoA ligase, the in situ assembled membranes were spontaneously modified with protein. Our strategy of coupling gene expression with membrane lipid synthesis in a one-pot fashion could facilitate the generation of proteoliposomes and brings us closer to the bottom-up generation of synthetic cells using recombinant synthetic biology platforms.

synthetic biology↗

bioRxiv2020

Light-activated tetrazines enable live-cell spatiotemporal control of bioorthogonal reactions

Bioorthogonal ligations encompass coupling chemistries that have considerable utility in living systems.1-3 Among the numerous bioorthogonal chemistries described to date, cycloaddition reactions between tetrazines and strained dienophiles are widely used in proteome, lipid, and glycan labeling due to their extremely rapid kinetics.4,5 In addition, a variety of functional groups can be released after the cycloaddition reaction,6,7 and drug delivery triggered by in vivo tetrazine ligation8 is in human phase I clinical trials.9 While applications of tetrazine ligations are growing in academia and industry, it has so far not been possible to control this chemistry to achieve the high degrees of spatial and temporal precision necessary for modifying mammalian cells with single-cell resolution. Here we demonstrate visible light-activated formation of tetrazines from photocaged dihydrotetrazines, which enables live-cell spatiotemporal control of rapid biorthogonal cycloaddition reactions between tetrazines and dienophiles such as trans-cyclooctenes (TCOs). Photocaged dihydrotetrazines are stable in conditions that normally degrade tetrazines, enabling efficient early-stage incorporation of bioorthogonal handles into biomolecules such as peptides. Photocaged dihydrotetrazines allow the use of non-toxic visible light to trigger tetrazine ligations on live mammalian cells. By tagging reactive phospholipids with fluorophores, we demonstrate modification of HeLa cell membranes with single-cell spatial resolution. Finally, we show that photo-triggered therapy is possible by coupling tetrazine photoactivation with strategies that uncage prodrugs in response to tetrazine ligation, opening up new methods for photopharmacology and precision drug delivery using bioorthogonal chemistry.

biochemistry↗

bioRxiv2020

A Small Molecule Fluorogenic Probe for the Detection of Sphingosine in Living Cells

ABSTRACTThe single-chained sphingolipid sphingosine is an essential structural lipid and signaling molecule. Abnormal sphingosine metabolism is observed in several diseases, including cancer, diabetes, and Alzheimer’s. Despite its biological importance, there are a lack of tools for detecting sphingosine in living cells. This is likely due to the broader challenge of developing highly selective and live-cell compatible affinity probes for hydrophobic lipid species. In this work, we have developed a small molecule fluorescent turn-on probe for labeling sphingosine in living cells. This probe utilizes a selective reaction between sphingosine and salicylaldehyde esters to fluorescently label sphingosine molecules. We demonstrate that this probe exhibits a dose-dependent response to sphingosine and is able to detect endogenous pools of sphingosine. Using our probe, we successfully detected sphingosine accumulation in live Niemann-Pick type C1 (NPC1) patient cells, a lipid transport disorder in which increased sphingosine mediates disease progression. This work provides a simple and accessible method for the detection of sphingosine and should facilitate study of this critical signaling lipid in biology and disease.Competing Interest StatementThe authors have declared no competing interest.View Full Text

biochemistry↗

bioRxiv2020

Enzymatic RNA Biotinylation for Affinity Purification and Identification of RNA-protein Interactions

Throughout their cellular lifetime, RNA transcripts are bound to proteins, playing crucial roles in RNA metabolism, trafficking, and function. Despite the importance of these interactions, identifying the proteins that interact with an RNA of interest in mammalian cells represents a major challenge in RNA biology. Leveraging the ability to site-specifically and covalently label an RNA of interest using E. Coli tRNA guanine transglycosylase and an unnatural nucleobase substrate, we establish the identification of RNA-protein interactions and the selective enrichment of cellular RNA in mammalian systems. We demonstrate the utility of this approach through the identification of known binding partners of 7SK snRNA via mass spectrometry. Through a minimal 4-nucleotide mutation of the long noncoding RNA HOTAIR, enzymatic biotinylation enables identification putative HOTAIR binding partners in MCF7 breast cancer cells that suggest new potential pathways for oncogenic function. Furthermore, using RNA sequencing and qPCR, we establish that an engineered enzyme variant achieves high levels of labeling selectivity against the human transcriptome allowing for 145-fold enrichment of cellular RNA directly from mammalian cell lysates. The flexibility and breadth of this approach suggests that this system could be routinely applied to the functional characterization of RNA, greatly expanding the toolbox available for studying mammalian RNA biology.

biochemistry↗

bioRxiv2020

Multiplexed photo-activation of mRNA with single-cell resolution

We demonstrate sequential optical activation of two types of mRNAs in the same mammalian cell through the sequential photocleavage of small molecule caging groups ( photo-cages) tethered to the 5' untranslated region (5'-UTR) of an mRNA. Synthetic photo-cages were conjugated onto target mRNA using RNA-TAG, an enzymatic site-specific RNA modification technique. Translation of mRNA was severely reduced upon conjugation of the photo-cages onto the 5'-UTR. However, subsequent photo-release of the cages from the mRNA transcript triggered activation of translation with single-cell spatiotemporal resolution. To achieve sequential photo-activation of two mRNAs in the same cell, we synthesized a pair of photo-cages which can be selectively cleaved from mRNA upon photo-irradiation with different wavelengths of light. Sequential photo-activation of two mRNAs enabled precise optical control of translation of two unique transcripts. We believe that this modular approach to precisely and rapidly control gene expression will serve as a powerful tool in future biological studies that require controlling translation of multiple transcripts with high spatiotemporal resolution.

biochemistry↗

bioRxiv2020

Lipid Sponge Droplets as Programmable Synthetic Organelles

Living cells segregate molecules and reactions in various subcellular compartments known as organelles. Spatial organization is likely essential for expanding the biochemical functions of synthetic reaction systems, including artificial cells. Many studies have attempted to mimic organelle functions using lamellar membrane-bound vesicles. However, vesicles typically suffer from highly limited transport across the membranes and an inability to mimic the dense membrane networks typically found in organelles such as the endoplasmic reticulum. Here we describe programmable synthetic organelles based on highly stable nonlamellar sponge phase droplets that spontaneously assemble from a single-chain galactolipid and non-ionic detergents. Due to their nanoporous structure, lipid sponge droplets readily exchange materials with the surrounding environment. In addition, the sponge phase contains a dense network of lipid bilayers and nanometric aqueous channels, which allows different classes of molecules to partition based on their size, polarity, and specific binding motifs. The sequestration of biologically relevant macromolecules can be programmed by the addition of suitably functionalized amphiphiles to the droplets. We demonstrate that droplets can harbor functional soluble and transmembrane proteins, allowing for the co-localization and concentration of enzymes and substrates to enhance reaction rates. Droplets protect bound proteins from proteases, and these interactions can be engineered to be reversible and optically controlled. Our results show that lipid sponge droplets permit the facile integration of membrane-rich environments and self-assembling spatial organization with biochemical reaction systems. Significance statementOrganelles spatially and temporally orchestrate biochemical reactions in a cell to a degree of precision that is still unattainable in synthetic reaction systems. Additionally, organelles such as the endoplasmic reticulum (ER) contain highly interconnected and dense membrane networks that provide large reaction spaces for both transmembrane and soluble enzymes. We present lipid sponge droplets to emulate the functions of organelles such as the ER. We demonstrate that lipid sponge droplets can be programmed to internally concentrate specific proteins, host and accelerate biochemical transformations, and to rapidly and reversibly sequester and release proteins to control enzymatic reactions. The self-assembled and programmable nature of lipid sponge droplets will facilitate the integration of complex functions for bottom up synthetic biology.

synthetic biology↗

bioRxiv2019

Enzyme-free synthesis of natural phospholipids in water

All living organisms synthesize phospholipids as the primary constituent of their cell membranes. While phospholipids can spontaneously self-assemble in water to form membrane-bound vesicles, their aqueous synthesis requires pre-existing membrane-embedded enzymes. This limitation has led to models in which the first cells used simpler types of membrane building blocks and has hampered integration of phospholipid synthesis into artificial cells. Here we demonstrate that a combination of ion pairing and self-assembly of reactants allows high-yielding synthesis of cellular phospholipids in water. Acylation of 2-lysophospholipids using cationic thioesters occurs in mildly alkaline solutions resulting in the formation of cell-like membranes. A variety of membrane-forming natural phospholipids can be synthesized. Membrane formation takes place in water from natural alkaline sources, such as soda lakes and hydrothermal oceanic vents. When formed vesicles are transferred to more acidic solutions, electrochemical proton gradients are spontaneously established and maintained.

biochemistry↗

bioRxiv2019

Laccase-mediated catalyzed fluorescent reporter deposition for live cell imaging

Catalyzed reporter deposition (CARD) is a widely established method for labeling biological samples analyzed using microscopy. Horseradish peroxidase, commonly used in CARD to amplify reporter signals, requires the addition of hydrogen peroxide which may perturb samples used in live-cell microscopy. Herein we describe an alternative method of performing CARD using a laccase enzyme, which does not require exogenous hydrogen peroxide. Laccase is an oxidative enzyme which can carry out single-electron oxidations of phenols and related compounds by reducing molecular oxygen. We demonstrate proof-of-concept for this technique through the non-targeted covalent labeling of bovine serum albumin using a fluorescently-labeled ferulic acid derivative as the laccase reporter substrate. We further demonstrate the viability of this approach by performing live-cell CARD with an antibody-conjugated laccase against a surface bound target. CARD using laccase produces an amplified fluorescence signal by labeling cells without the need for exogenous hydrogen peroxide.

biochemistry↗

bioRxiv2019

Prevalence, awareness and control of hypertension in Malaysia 1980 - 2017: A Systematic Review and Meta-Analysis

BackgroundHypertension is a common public health problem worldwide and is a well-known risk factor for increased risk of cardiovascular diseases, contributing to high morbidity and mortality. However, there is no systematic review and meta-analysis that has been done in a multi-ethnic population like Malaysia. This systematic review aims to determine the trend in prevalence, awareness and control of hypertension in Malaysia.\n\nMethodsSystematic searches were conducted in PubMed, Scopus, Ovid, Cumulative Index to Nursing and Allied Health Literature, Malaysian Medical Repository and Malaysia Citation Index published between 1980 and 2017. All original articles in English were included. Studies included were those on adults aged 18 years and above. Studies of prevalence in children and adolescents and pregnancy related hypertension were excluded. Two authors independently reviewed the studies, carried out data extraction and performed quality assessment. Heterogeneity between studies and publication bias was assessed and effect size was pooled by the random effect model.\n\nResultsFifty-six studies with a total of 241,796 subjects were included. The prevalence of hypertension throughout Malaysia varied (I2 = 99.3%). The overall pooled prevalence of hypertension over the past 4 decades was 28.2% in adults aged 18 years and older (95% CI: 26.1 - 33.3) and the prevalence in those 30 years and older was 40.0% (95% CI: 35.3-44.8).\n\nFor subgroup analysis, the prevalence of hypertension in male aged 18 and above was 31.4% (95% CI: 26.5 - 36.2) and 27.8% in female (95% CI: 20.7 - 34.9). The prevalence of hypertension among the ethnic groups aged 18 years and above were 37.3% in Malays (95% CI: 32.9 - 41.7); 36.4% in Chinese (95% CI 31.6 - 41.2) and 34.8% in Indians (95% CI: 31.2-38.4). The prevalence of hypertension was the lowest in the 1980s (16.2%, 95% CI: 13.4-19.0%), increases up to 36.8% in the 1990s (95% CI: 6.1-67.5), then came down to 28.7% (95% CI: 21.7-35.8) in the 2000s and 29.2% (95% CI: 24.0-34.4) in the 2010s. The prevalence of awareness was 38.7% (95% CI: 31.7 - 45.8) whereas the control of hypertension of those on treatment was 33.3% (95% CI: 28.4 - 38.2).\n\nConclusionThree in 10 adults aged 18 years old and above have hypertension, whereas four in 10 adults aged 30 years old and above have hypertension. Four out of 10 are aware of their hypertension status and only one-third of them who were under treatment achieved control of their hypertension. Concerted efforts by policymakers and healthcare professionals to improve the awareness and control of hypertension should be of high priority.

epidemiology↗

bioRxiv2018

Communication and quorum sensing in non-living mimics of eukaryotic cells

Cells in tissues or biofilms communicate with one another through chemical and mechanical signals to coordinate collective behaviors. Non-living cell mimics provide simplified models of natural systems, however, it has remained challenging to implement communication capabilities comparable to living cells. Here we present a porous artificial cell-mimic containing a nucleus-like DNA-hydrogel compartment that is able to express and display proteins, and communicate with neighboring cell-mimics through diffusive protein signals. We show that communication between cell-mimics allows distribution of tasks, quorum sensing, and cellular differentiation according to local environment. Cell-mimics can be manufactured in large quantities, easily stored, chemically modified, and spatially organized into diffusively connected tissue-like arrangements, offering a means for studying communication in large ensembles of artificial cells.

synthetic biology↗