Small amphiphilic DNA for programmable transmembrane signaling and amplification

Small amphiphilic DNA for programmable transmembrane signaling and amplification | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results Small amphiphilic DNA for programmable transmembrane signaling and amplification View ORCID Profile Ranjan Sasmal , View ORCID Profile Saanya Yadav , View ORCID Profile Gde Bimananda Mahardika Wisna , View ORCID Profile Nirbhik Acharya , View ORCID Profile Carter Swanson , Hao Yan , View ORCID Profile Himanshu Joshi , View ORCID Profile Rizal F. Hariadi doi: https://doi.org/10.1101/2025.10.29.685379 Ranjan Sasmal a Center for Molecular Design and Biomimetics at the Biodesign Institute, Arizona State University , Tempe, Arizona, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Ranjan Sasmal For correspondence: rsasmal{at}asu.edu rhariadi{at}asu.edu Saanya Yadav b Department of Biotechnology, Indian Institute of Technology , Hyderabad, India Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Saanya Yadav Gde Bimananda Mahardika Wisna a Center for Molecular Design and Biomimetics at the Biodesign Institute, Arizona State University , Tempe, Arizona, USA c Department of Physics, Arizona State University , Tempe, Arizona, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Gde Bimananda Mahardika Wisna Nirbhik Acharya a Center for Molecular Design and Biomimetics at the Biodesign Institute, Arizona State University , Tempe, Arizona, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Nirbhik Acharya Carter Swanson a Center for Molecular Design and Biomimetics at the Biodesign Institute, Arizona State University , Tempe, Arizona, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Carter Swanson Hao Yan a Center for Molecular Design and Biomimetics at the Biodesign Institute, Arizona State University , Tempe, Arizona, USA d School of Molecular Sciences, Arizona State University , Tempe, Arizona, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Himanshu Joshi b Department of Biotechnology, Indian Institute of Technology , Hyderabad, India Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Himanshu Joshi Rizal F. Hariadi a Center for Molecular Design and Biomimetics at the Biodesign Institute, Arizona State University , Tempe, Arizona, USA c Department of Physics, Arizona State University , Tempe, Arizona, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Rizal F. Hariadi For correspondence: rsasmal{at}asu.edu rhariadi{at}asu.edu Abstract Full Text Info/History Metrics Preview PDF Abstract Transmembrane proteins such as G-protein coupled receptors (GPCRs) transmit molecular signals across lipid bilayers through stimulus-responsive allosteric mechanisms, where extracellular ligand binding induces G-protein dissociation to initiate downstream signaling. Mimicking such specific signal transduction pathway with DNA nanostructures has remained challenging due to the incompatibility between hydrophilic DNA and hydrophobic membranes, and the difficulty of engineering allosteric DNA reactions across lipid bilayers. Here, we overcome these limitations by exploiting DNA Hybridization Across Lipid for Optical Signaling (HALOS) using an amphiphilic DNA hairpin comprising toehold for recognition, stem for stability, loop, and cholesterols for transmembrane anchoring. Upon binding of a single-stranded nucleic acid target stimuli, strand invasion through the toehold initiates conformational switching in HALOS, enabling signal transduction across membrane. Experimentally, we demonstrate that DNA hybridization across lipid membrane can occur, contradicting the prevailing view that DNA hybridization cannot proceed through hydrophobic barriers. All-atom molecular dynamics (MD) simulations reveal that cholesterol tags stabilize the DNA stem within the bilayer, preserving the hairpin structure necessary for transmembrane signaling. By combining the HALOS with a non-enzymatic isothermal hybridization chain reaction (HCR), we establish a platform that enables intracellular nucleic acid target detection and amplified fluorescent reporting from outside synthetic vesicles and live mammalian cells achieving nanomolar sensitivity. HALOS expands the toolkit for membrane-integrated DNA nanotechnology and opens avenues for lysis-free diagnostics, synthetic cell and biology, and targeted therapeutic activation. Key words: Amphiphilic DNA Nanosensor, transmembrane signaling, HCR amplification, intracellular RNA sensing, molecular dynamics, next–generation HCR. Introduction N on-invasive detection of intracellular molecules remains a fundamental challenge in biology and medicine, as it requires transmembrane signal transduction while maintaining cellular integrity. 1 – 3 Natural receptors such as G-protein-coupled receptors (GPCRs) binds to extracellular ligands and converting them into intracellular signals through tightly controlled signaling cascades. 4 – 7 These membrane receptors regulate diverse physiological processes, making them major therapeutic targets. 4 , 8 – 10 However, their specificity is evolutionarily constrained by native ligands, limiting their adaptability to novel targets. Rationally reprogramming GPCRs presents significant challenges, including structural complexity and potential loss of function. 11 Engineering synthetic transmembrane signal transducers requires solving significant challenges in chemical design: achieving selective molecular recognition, coupling binding events to conformational changes spanning lipid bilayers, and maintaining structural integrity in complex biological environments. Synthetic GPCR-mimics with programmable intracellular target recognition could thus enable a new class of biosensors for real-time cellular monitoring and engineering–capabilities beyond reach of existing molecular devices. Chemical strategies for synthetic transmembrane signaling have employed diverse molecular architectures, including engineered peptides and proteins 12 – 17 , lipid and small molecule assemblies. 18 – 20 Designing protein-based GPCR mimics for novel targets is often hindered by laborious optimization and vulnerability to instability or immunogenicity in biological environments. 21 – 23 While lipid-based systems excel at membrane incorporation but typically fail to achieve programmability and robust transduction, small molecule approaches lack both the structural complexity necessary for selective recognition and the capacity for signal amplification. 20 , 24 These limitations highlight the need for alternative molecular architectures that combine chemical stability, programmable recognition through well-understood chemical principles, and rational design approaches. Addressing these challenges, DNA nanotechnology has emerged as a unique chemical platform that integrates programmable molecular recognition, chemical stability, and nanoscale architectural control, making it especially suited for engineering synthetic GPCR mimics and membrane-spanning functional nanodevices. 25 – 30 Due to its biocompatibility, nanoscale precision, structural programmability, and chemical flexibility, DNA nanostructures have been widely explored in biosensing, tissue engineering, disease therapy, and in vivo imaging. 31 – 34 Additionally, versatile DNA assemblies spanning lipid bilayers have served as synthetic receptors and channels, enabling controlled transport of diverse cargo such as ions 35 – 38 , small molecules 39 , 40 , polymers 41 , DNA 42 , 43 , proteins and lipids 44 , 45 . Also, stimuli-responsive DNA nanopores with gated architectures was achieved mimicking natural ion channel by reversible switching between open and closed states. 46 – 48 However, most DNA membrane systems have focused on molecular transport rather than non-invasive signal transduction process. Designing a functional transmembrane signal transducer requires overcoming several challenges including stable membrane integration through hydrophobic modifications that preserve bilayer integrity, coupling molecular recognition to conformational changes that span the bilayer, and amplifying signals into optically detectable outputs. Recently, DNA-based nanodevices achieved GPCRs-like signal transduction across the membrane, notably without physically transporting signaling molecules. 49 – 52 Despite advancements, current DNA-based nanodevices remain limited by pH dependency, reliance on complex delivery methods like vesicle fusion for transmembrane nanodevice formation, and ineffective performance in live cells. Most critically, they lack the sensitivity and signal-to-noise ratio to detect low-abundance intracellular nucleic acid targets in synthetic and living environments. We report Hybridization Across Lipid for Optical Signaling (HALOS), an amphiphilic DNA-based synthetic GPCR-mimic that enables detection of sequence–specific intracellular nucleic acid targets and transmembrane signaling without perturbing membrane integrity. HALOS integrates cholesterol–mediated robust membrane anchoring with programmable hairpin DNA and ∼10 × signal amplification using non–enzymatic hybridization chain reaction (HCR) strategy, achieving nanomolar sensitivity for target nucleic acid detection in live cells. Unlike previous DNA-based transmembrane signaling devices 50 , 52 , which often require pH modulation, vesicle fusion or complex delivery strategies for their functions, HALOS enables direct functional integration within native cellular membranes. Additionally, its single-hairpin design inherently circumvents stoichiometric assembly errors and post-formation purification typical of multi-strand DNA devices. The working principle employs a membrane-spanning structural changes in HALOS from hairpin-to-linear conformational switch upon coupling intracellular targets and subsequent extracellular reporter binding to open hairpin domain, enabling signal transduction and amplification across lipid bilayer, suitable for diagnostic applications. Molecular dynamics (MD) simulation reveals inherent stability of membrane spanning HALOS and its intermediate states upon conformational switching, while experimental validation demonstrates nanomolar target detection sensitivity. We further elucidate the mechanistic characterization of HALOS-mediated signal transduction to probe the extend of DNA hybridization across membrane and next–generation signal amplification strategies. Our work establishes a new class of programmable synthetic GPCR mimics with the sensitivity and robustness required for cellular diagnostics, synthetic and chemical biology, and therapeutic monitoring. Results HALOS design principle for transmembrane signaling Inspired by GPCR signaling pathway where an extracellular ligand binding triggers an allosteric conformational change relaying the signal across lipid bilayer and activates the intracellular biological reaction cascades 5 , 7 ( Fig. 1(a) ), we designed a transmembrane signal transducer using amphiphilic DNA hairpin system ( Fig. 1(b) ). HALOS consists of a toehold domain (a) for programmable target recognition, a membrane–stabilizing stem (b–b*) with dual cholesterol anchors positioned 180° apart (5 bp) for optimal membrane insertion, and a stability–optimized loop (c) that maintains structural integrity in the lipid environment ( Fig. 1(b) ). Two cholesterol anchors were selected over other lipid anchors due to superior membrane affinity, insertion and stability. 45 , 53 , 54 Intracellular target recognition and conformational changes in HALOS externalize the non–cholesterol domain (b*), enabling fluorescently labeled reporter binding and generating signal outside the cells. Download figure Open in new tab Fig. 1. Structural optimization with stable membrane integration and functional characterization of HALOS through MD simulation. (a) Schematic of GPCR–mediated signaling across the membrane: extracellular ligand binding, conformational switching and intracellular signaling cascades. (b) HALOS as synthetic GPCR mimic: anchored within lipid bilayer via two cholesterol moieties, displaying a 10–nt target recognition domain (a), optimized 14–31 bp stem (b–b*) and a 10–nt loop. (c) Reaction graph of GPCR–like signal transduction using membrane anchored HALOS: intracellular target binding triggers conformational switching, enabling extracellular reporter recruitment and signal amplification on membrane. (d) Systemic stepwise illustration of HALOS–mediated signal transduction: membrane attachment, orientation alignment, toehold–mediated target recognition, conformational switching, and fluorescence signal amplification via HCR on membrane. (e) Design optimization rationale: shorter stems cause spontaneous opening and false signals by labeling exposed domain (b*), requiring systematic length optimization. (f) HALOS 14 and HALOS 31 comparison in axial membrane orientation reveals stability differences. (g–h) Confocal microscopy demonstrating HALOS 31 superiority with ∼ 99% specificity in GUVs ( g ) and HEK293T cells ( h ), while HALOS 14 shows false signaling. Flow cytometry analysis of Cy5 signals on GUVs and cell membranes anchored with HALOS 14 and HALOS 31 (n=10,000 cells). (i–l) Snapshots of the HALOS 31 for 1 µs MD simulation trajectory in various environments; side (i) and top (ii) views of HALOS 31 in aqueous media ( i ), lateral ( j ), axial ( k ) and target bound HALOS ( l ) on membrane. Distribution of cumulative hydrogen bonds between the nitrogenous bases in the double–stranded stem of HALOS (iii) and cholesterol angle–distribution (iv) obtained from the respective simulation trajectories. The DNA backbone is represented as tube while the nitrogenous bases are shown in sphere representation. The volume occupied by lipid molecules is shown in transparent white background. Water and ions are not shown for clarity. To summarize the transduction pathway, we depict membrane–spanning HALOS reaction graph using a nodal abstraction consisting a triangular input and circular output port ( Fig. 1(c) ). The state of each port is either accessible (open symbol) or inaccessible (filled symbol), depending on whether DNA recognition motifs are exposed or sequestered. 55 Recognition of a complementary sequence target nucleic acids by HALOS converts open to filled symbols in target recognition input port and subsequent DNA hybridization across membrane results in generation of open accessible output port for reporter binding on HALOS. A cascade of DNA chain reactions using metastable reporters with the open output port thus illustrates signal amplification on membrane. HALOS achieves the first synthetic outward transmembrane signaling mechanism, detecting intracellular targets and amplifying signals directly on membrane without requiring cell lysis or genetic modification ( Fig. 1(d) ). The proposed pathway initiates with cholesterol–mediated membrane anchoring in a lateral orientation, followed by insertion via axial reorientation, positioning the toehold domain for intracellular target access. Upon recognition of sequence–specific (a*–b*) Cy3 labeled targets (T–Cy3), toehold–mediated strand displacement initiates DNA hybridization and hairpin opening across the membrane, externalizing the reporter binding domain (b*) and enabling Cy5–labeled reporter (R–Cy5) recruitment. Critically, signal amplification occurs through membrane–localized hybridization chain reaction (HCR) on b*, where alternating strand–exchange polymerization between metastable hairpin monomers (H1 and H2) creates a robust fluorescent polymer directly on the membrane surface. This integrated strategy enables sensitive detection of intracellular targets while maintaining membrane integrity, establishing a new paradigm for non–invasive cellular sensing. HALOS transmembrane stability is dictated by the stem length Accurate transmembrane signaling by HALOS requires a hairpin stem that remains stable within the lipid bilayer, resisting hydrophobic interactions, membrane fluctuations, and base–pair disruption. Insufficient stem stability can lead to spontaneous opening during membrane anchoring and insertion, resulting in false–positive R–Cy5 labeling ( Fig. 1(e) ). To address this, we evaluated the membrane stability of hairpin stems from 14 to 31 bp each with dual mid–stem cholesterol anchors using all–atom MD simulations ( Fig. 1(f) , SFig. 1 ). MD simulations revealed that shorter stems in HALOS with 14 bp (HALOS 14) had a higher frequency of base–pair breaks compared to longer stems such as HALOS 31 in both aqueous and with membrane, demonstrating that increased stem length confers greater structural integrity on the membrane. Systematic experimental validation on synthetic cells namely, giant unilamellar vesicles (GUVs) prepared using POPC lipids (1–palmitoyl–2–oleoyl–glycero–3–phosphocholine) and live HEK293T cells confirmed that HALOS 31 eliminates false–positive signaling while maintaining target responsiveness, establishing the design parameters required for reliable cellular signaling applications. In the absence of target DNA (GUVs filled with ATTO488 dyes only), we isolated the effects of stem–stability by monitoring R–Cy5 binding to the exposed b* domain on HALOS as an indicator of spontaneous hairpin destabilization ( Fig. 1(e) ). Consistent with stem instability, HALOS constructs with 14 to 28 base pair stems produced significant Cy5–positive membrane labeling on ATTO488 GUVs, diminishing from 14 bp to 28 bp as stem length increased (( Fig. 1(g) left, SFig. 2 ). Critically, HALOS 31 exhibited no detectable R–Cy5 labeling ( Fig. 1(g) right), a finding corroborated by quantitative flow cytometry, which revealed negligible Cy5 fluorescence and confirmed its superior structural stability ( Fig. 1(g) below, S Fig. 2 ). Download figure Open in new tab Fig. 2. HALOS 31 achieves enhanced signal transduction with comprehensive specificity validation. (a) Negative controls demonstrate specificity in signal transduction: abstraction showing absence of HALOS 31 and reporters prevents target recruitment and reporter signaling (i–iii). External complementary strands quench free targets to suppress false positive target recognition. Confocal microscopy showing no Cy3 labeling without HALOS 31, while HALOS 31 presence enables target binding on membrane. Minimal Cy5 signal (374 ± 280 a.u., n=10,000 cells) confirms low background transduction. (b) Positive signal transduction validation: abstraction and schematic demonstrating target binding triggers HALOS 31 conformational change, exposing open hairpin domain (b*) for reporter recognition. Confocal microscopy revealing co–localized Cy3 and Cy5 signals with elevated flow cytometry signal (1,289 ± 881, n=10,000 cells) confirm efficient transmembrane signal transfer. (c) Scramble controls validate sequence specificity: scrambled targets prevent HALOS 31 recognition and conformational switching, blocking Cy5 labeling. Scrambled reporters fail to bind exposed hairpin despite successful target engagement. Confocal microscopy showing no membrane fluorescence for scrambled targets, while scrambled reporters display Cy3 but prevent Cy5 signaling. Flow cytometry confirms minimal Cy5 signals for both scrambled target (160±138 a.u., n=10,000 cells) and reporter (170±142 a.u.) controls (n=10,000 cells each). On live HEK293T cells, HALOS variants with stem lengths of 25 bp or longer demonstrated robust stability, with HALOS 31 selected as the optimal construct for subsequent studies due to its consistent performance across systems. R–Cy5 fluorescence on HEK293T cells revealed spontaneous destabilization exclusively in hairpin constructs with shorter 14–21 bp stems ( Fig. 1(h) , SFig. 2 ). In contrast, HALOS variants with longer stems (25–31 bp) remained unlabeled (∼ 1% Cy5 positive cells), indicating stable HALOS stem within cell membrane. Quantitative flow cytometry was concordant, detecting significant Cy5 signal for HALOS 14 and HALOS 21 only (∼ 99% and ∼ 15% positive cells respectively) on the membrane ( Fig. 1(h) , SFig. 2 ). The observed difference in stability thresholds between GUVs (stable at ≥ 31 bp) and live cells (stable at ≥ 25 bp) likely reflects variations in membrane composition and mechanical properties. Based on these comprehensive findings, HALOS 31 was selected for all subsequent studies, providing an optimized balance of structural integrity and minimal false–positive signal generation in both synthetic and cellular environments. Molecular dynamics reveal membrane anchoring and transmembrane stability of HALOS All–atom MD simulations revealed that HALOS 31 adopts a highly stable axial orientation within the lipid bilayer, and target binding triggers a thermodynamically driven expulsion of the non–cholesterol stem (b*) to the aqueous phase, initiating signal transduction. Simulations were conducted to characterize HALOS interactions with the lipid bilayer, capturing diverse molecular orientations and structural intermediates to analyze orientation, stability, and signal transduction. We systematically compared HALOS stability across various states including aqueous, lateral, axial, intermediate, open conformations using hydrogen–bond analysis and quantified angle between two cholesterol anchors ( Fig. 1(i–l) , SFig. 3 ). Side and top views from post MD simulation snapshots demonstrated that HALOS 31 stably resides within the lipid bilayer while maintaining overall conformational integrity. Over 1 µs of simulation, the DNA hairpin exhibited substantial structural fluctuations, primarily in the flexible loop and single–stranded toehold domains. Notably, without the lipid bilayer, the double–stranded stem of HALOS remained largely stable, however, cholesterol moieties tended to stack and intercalate bases to avoid the aqueous environment, resulting in significant helical distortion. Download figure Open in new tab Fig. 3. Bulky triangle reporter and optimized membrane fluidity enable leakless, target–specific signal transduction in mixed GUV populations. (a) Triangular reporter design demonstrating two complementary DNA strands forming 7–nm triangle with Cy5 and biotin labels, enhanced by streptavidin (5 nm) to prevent leakage from misfolded reporters. (b) Bulky SA–TrCy5 reporter labeling to target–recognized HALOS 31, preventing nanopore–mediated leakage across membrane. (c) Schematic of signal transduction validation from target–specific GUVs using SA–TrCy5 in mixed populations while excluding controls (ATTO488). (d) Confocal microscopy demonstrates selective signaling from T–Cy3–encapsulated GUVs showing membrane labeling while controls lack Cy3 and Cy5 signals, confirming zero false positives. (e) Quantitative detection analysis: T–Cy3 GUVs showed (52 ± 5%) with both Cy3 and Cy5 rings and (3 ± 2%) partial Cy3 recognition but Cy5 rings, revealing signal transduction in both the populations. The remaining (39 ± 5%) were either unrecognized or recognized (6 ± 2%) but showed no Cy5 signal (n=350 GUVs). ATTO488 GUVs (control) showing very minimal Cy3 and Cy5 labeling (2 ± 1%) on the membrane, suggesting negligible non–specific activation of HALOS from outside T–Cy3 (n=420 GUVs). (f) Systematic membrane fluidity variation and transmembrane signaling optimization reveals significant enhancement from more rigid POPC (31 ± 5%) to more fluid EggPC+LPC (83 ± 4%), with intermediate compositions showing progressive improvement (EggPC 42 ± 5%, POPC+Chol 53 ± 5%, POPC+LPC 66 ± 5%; n=850 GUVs each). Error bars from bootstrapping analysis. Quantitative analysis of base–pair stability in HALOS confirmed that membrane incorporation significantly enhances structural integrity, with axial orientation providing superior stability. The time evolution of broken base pairs which are defined by hydrogen bond thresholds (A–T: <2 H–bonds; C–G: <3 H–bonds; distance <3.4 Å, angle <45 ° ), showed minimal disruption in axially anchored hairpins, while HALOS in aqueous solution exhibited increased base–pair breaks near the end of simulation ( SFig. 1 ). Hydrogen bond analysis confirmed HALOS possesses the greatest stability in the axial state (mean 77.2) compared to lateral (mean 69.2) and in solution (mean 61.8) ( Fig. 1(i–k) ). Contour length distributions further indicated that in aqueous or laterally oriented HALOS states was overstretched around hydrophobic modifications ( SFig. 3 ). Consistently, the root mean square fluctuations (RMSF) data showed that membrane incorporation stabilized overall HALOS structure, while hairpin stems in aqueous solution experienced substantial conformational fluctuations ( SFig. 3 ). The angle between HALOS cholesterol anchors critically determines stem integrity and membrane orientation, directly impacting target recognition function. A small angle (0–30 ° ) induces cholesterol stacking in aqueous and lateral membrane orientation, causing stem distortion and an unfavorable helical conformation ( Fig. 1(i-j) ). Conversely, a large angle (100–210°) promotes stable axial orientation, resulting in increased stability and positioning HALOS perpendicular to the membrane, thereby facilitating target recognition ( Fig. 1(k) ). MD simulations on target–bound HALOS in intermediate and open hairpin conformations confirm the feasibility of signal transduction by membrane–anchored HALOS. The increased number of hydrogen bonds (mean 87.9) compared to HALOS alone and a distinct large angle distribution between cholesterol anchors (60–150 ° ) confirm the stable residence of these constructs within the membrane ( Figs. 1(l) , SFig. 3 ). These conformational changes, characterized by the altered H–bond network and cholesterol anchor angle, facilitates the exposure of the b* arm to the aqueous phase, enabling R–Cy5 recruitment on opened HALOS domain and signal transduction across membrane. HALOS 31 enables specific transmembrane target detection and signal transduction in GUVs Following successful evaluation of HALOS 31 stability on the lipid membrane, we assessed its efficacy as a transmembrane sensor for target detection on GUVs. NUPACK design predictions 56 and gel electrophoresis confirmed the specificity of target recognition, hairpin opening, and subsequent reporter labeling by HALOS 31 in solution ( SFig. 4 ). To test membrane–embedded detection, T–Cy3 (41–nt) was encapsulated within POPC GUVs via electroformation 57 , validated by the formation of lipid spheres containing Cy3 dyes ( Fig. 2(a) left). To rigorously exclude false–positive signals generated from external target activated laterally anchored HALOS variants, the outside targets were eliminated through sequential centrifugation and washing steps, and by incubating GUVs with ∼ 20 × excess complementary DNA against target strand to quench residual external target activity ( SFig. 5 ). The nodal abstractions illustrated target recognition and signal transduction by membrane embedded HALOS 31 as indicated by changing open to filled symbols. Upon incubating these GUVs with HALOS 31, a rapid and pronounced Cy3 labeling appeared on the membranes, indicating efficient membrane insertion and target recognition ( Fig. 2(a) right, SFig. 5 ). A progressive decrease of Cy3 fluorescence inside the GUVs, alongside its accumulation at the inner leaflet, revealed the spatial redistribution of labeled targets, consistent with target recognition by HALOS 31. Quantification of sensor activity showed that (∼ 85 ± 5)% of GUVs exhibited distinct Cy3–rings within 90–120 minutes ( SFig. 5 ). Analysis of target recognition kinetics demonstrated heterogeneity in the rate of Cy3 accumulation at the membrane periphery over 120 minutes ( SFig. 6 ), likely reflecting variable lipid rafts (monolayer vs. multilayer), target encapsulation efficiencies based on GUVs size variations, and HALOS 31 population on individual GUVs. Notably, no target detection events were observed in the absence of HALOS 31 and lacking cholesterol moieties on HALOS 31 ( Fig. 2 (a) middle, SFig. 7 ), highlighting the essential role of cholesterol in HALOS design. Download figure Open in new tab Fig. 4. HALOS 31 enables lysis–free detection of cytosolic nucleic acids. (a) Schematic illustrating lysis–free detection of 100–nt long DNA targets encapsulated within GUVs, with signal transduction mediated by SA–TrCy5 labeling. (b) Confocal images reveal Cy3 and Cy5 membrane labeling for HALOS–mediated target recognition and signal transduction respectively. (c) Quantitative analysis displaying efficient signal transduction for three variants of extended DNA targets with varied position of 41–nt recognition domains: E1 (98 ± 1%), E2 (96 ± 2%), E3 (88 ± 3%) (n=200 GUVs each). (d–f) Schematic ( (d) ), confocal images ( (e) ), and quantitative analysis ( (f) ) of 41–nt 2 ′ –OMe modified RNA target detection (87 ± 4%) and signal transduction (58 ± 5%, n=90 GUVs), confirming capability of HALOS 31 for diverse intracellular nucleic acid detection. Download figure Open in new tab Fig. 5. HCR amplification on membrane achieves 8.2–fold signal enhancement in HALOS–mediated nucleic acid detection. (a) Abstraction graph of target–recognized HALOS initiating extracellular HCR using two metastable hairpins (H1, H2) for signal amplification. (b) Schematic of HALOS–mediated target recognition, generating initiator for HCR on membranes, triggering stepwise fluorescence signal amplification. (c) Confocal microscopy of GUV membranes demonstrates HCR–mediated signal amplification: no Cy5 signal in controls lacking HALOS 31 or targets, minimal signal with hairpin 1 alone, and strong amplification with both hairpins. Upper triangle images of Cy5 channel were shown with 5 brightness for visualization purposes. (d) Flow cytometry quantification reveals background corrected ∼ 4.2× amplification in GUVs: background levels without HALOS 31 (450 ± 394 a.u.) or targets (453 ± 279 a.u.), detectable signal with H1 (1058 ± 491 a.u.), and significantly higher with HCR amplification (3017 ± 1591 a.u., n=10,000 GUVs each). (e–f) Live A549 cells demonstrate intracellular RNA detection with HCR amplification. Confocal imaging shows Cy3 (RNA target recognition) and Cy5 (HCR amplification) membrane signals, with ∼ 6.9× enhancement over hairpin 1 alone (p<0.0001, n=60 cells). Controls lacking HALOS 31 or targets showed no membrane labeling. (g) Flow cytometry quantification on live HEK293T cells confirms ∼ 8.2× signal amplification: minimal background without HALOS 31 (168 ± 103 a.u.), without targets (296 ± 171 a.u.), moderate signal with hairpin 1 (360 ± 134 a.u.), and significantly higher with HCR (1739 ± 598 a.u., n=10,000 cells each). Download figure Open in new tab Fig. 6. Dual reporter labeling and proximity based ψ –HCR reveals DNA branch migration across the membrane. (a) Abstraction of spectrally distinct dual reporter labeling to T ′ –Cy3 recognized HALOS 31 on membrane. (b) Schematic of T ′ –Cy3 detection by HALOS 31, migration of extended targets across membrane and orthogonal dual reporter labeling to extended domain of target and HALOS open hairpin domain respectively, confirming complete DNA migration across membranes. (c) Confocal microscopy demonstrates individual labeling by SA–TrCy5 (red), SA–TrAF488 (blue), and simultaneous dual labeling when both reporters were applied, confirming absence of cross–reactivity. (d) Quantitative analysis of dual reporter membrane labeling shows specific binding: SA–TrCy5 alone (53 ± 3%), SA–TrAF488 alone (39 ± 4%), and both reporters simultaneously (53 ± 3% and 39 ± 4% respectively, n=150 GUVs each). (e) Abstraction of ψ –HCR on membrane by T ′ –Cy3–recognized HALOS 31. (f) Target extension and hairpin opening of HALOS 31 trigger formation of proximal split–initiator, enabling ψ –HCR amplification using metastable hairpin monomers (H1, H2). (g–h) Confocal microscopy demonstrate ψ –HCR–mediated signal enhancement on live A549 cell membrane ( g ). Controls lacking HALOS 31 or extended d* domain from 2 ′ –OMe-RNA target (41–nt) showed no membrane Cy5 signal. HCR signal amplification was observed with extended target (60–nt 2 ′ –OMe-RNA) transfected cells, achieving ∼ 5.4× amplification over hairpin 1 alone ( h , p<0.0001, n=50 cells). (i) Flow cytometry quantification on live HEK293T cells confirms ∼ 2.6× signal amplification: minimal background without HALOS 31 (1800 ± 1300 a.u.) or targets (2900 ± 1800 a.u.), moderate signal with hairpin 1 (63004 ± 2117 a.u.), and significantly higher with ψ –HCR (14000 ± 7000 a.u., n=10,000 cells each). To assess the signal transduction capability of HALOS 31 as a synthetic GPCR analogue, we triggered its conformational switch with T–Cy3 detection (green), enabling R–Cy5 labeling (red) to the exposed extracellular domain (b*) from outside, indicating successful transmembrane signaling ( Fig. 2(b) ). Confocal microscopy showed both Cy3 and Cy5 labeling only in GUVs containing HALOS 31, with controls lacking HALOS 31 and T–Cy3 displaying no Cy5 signal ( Fig. 2(a–b) , SFig. 7 ). Quantitative flow cytometry analysis demonstrated a significant increase in mean Cy5 fluorescence on GUVs containing HALOS 31 (1289 ± 881) compared to its controls (374 ± 280), confirming efficient and specific transmembrane signal transduction ( Fig. 2(a–b) below). Consistently, Cy3 vs Cy5 intensity plots show that ∼ 27% of T–Cy3 GUVs with HALOS 31 display overlapping membrane labeling, compared to ∼ 1.8% without HALOS 31 ( SFig. 7 ), highlighting the efficiency of signal transduction. To mimic canonical GPCR signaling from outside to inside the membrane, we performed an analogous assay on GUVs. Since HALOS 31 inserts to lipid membrane in an unbiased orientation (Toehold–in and toehold–out) during membrane anchoring, a fraction remains accessible for external target recognition ( SFig. 8 ). Upon externally added T–Cy3 binding, hairpin opening was initiated from the outside, resulting in conformational changes to inside the GUVs, as evidenced by successful labeling with pre–encapsulated R–Cy5 GUVs ( SFig. 8 ). No signal transduction was observed in the absence of external T–Cy3 ( SFig. 8 ). The live–cell compatibility of HALOS 31 along with T–Cy3 and R–Cy5 DNA were also evaluated by conducting HALOS–mediated signal transduction on live HEK293T cell membrane through externally triggered T–Cy3 recognition followed by R–Cy5 labeling ( SFig. 8 ). Interestingly, HALOS 31 exhibited high target selectivity, as mismatched DNA targets (Scr. T–Cy3) did not induce membrane Cy3 and Cy5 labeling ( Fig. 2(c) left, SFig. 9 ). These results collectively established HALOS 31 as a robust and specific transmembrane sensor. Similarly, to assess the specificity of R–Cy5 labeling, we tested mismatched reporter sequences (Scr. R–Cy5) on GUVs with correctly targeted open state of HALOS and observed no non–specific Cy5 membrane labeling by confocal microscopy ( Fig. 2(c) right, SFig. 9 ). Flow cytometry further confirmed negligible Cy5 labeling with both Scr. T–Cy3 (0.6%) and Scr. R–Cy5 (0.3%; Fig. 2(c) below, SFig. 9 ). Next–generation bulkier reporter design enables ‘leakless’ signal transduction across membrane While monitoring HALOS–mediated signal transduction across GUVs ( Fig. 2(b) ), an unexpected R–Cy5 leakage was detected partially in certain GUVs populations ( SFig. 7 ). This leakage likely resulted from transient nanopore formation by DNA sensor duplexes with dual cholesterol anchors 38 , 49 , 58 , allowing 13–nt ssDNA R–Cy5 (radius of gyration ∼ 1.7 nm, supplementary methods) to diffuse through the membrane during HALOS conformation switch. This diffusion could lead to false positives by labeling partially anchored T–Cy3 within the GUV inner lumen ( SFig. 10 ). Extending R–Cy5 ssDNA strand length to 31-nt reduced the leakage but did not completely eliminate this undesired effect. To achieve ‘leakless’ signal transduction, we designed a bulky biotinylated triangle–shaped reporter (Tr, ∼ 7 nm edge) with a 21–nt labeling domain ( Fig. 3(a) , SFig. 10 ). Labeling the open HALOS state on the exterior membrane with Tr reporters effectively extrudes them outward, effectively preventing its leakage through nanopores ( Fig. 3(b) ). Gel electrophoresis confirmed Tr formation and its specific in solution labeling with T–Cy3 anchored HALOS 31 ( SFig. 10 ). Using a streptavidin–loaded TrCy5 reporter (SA–TrCy5) resulted in a significant reduction of R–Cy5 leakage within GUVs ( Fig. 3(c–d) , SFig. 10 ), as the larger SA (∼ 5 nm) increases the reporter size and sterically hinders the leakage of misfolded TrCy5. Lysis–free detection of specific intracellular nucleic acid targets remains a significant challenge in biological research. To enable this, we performed signal transduction using a mix population of T–Cy3 encapsulated and non–target control (ATTO488) GUVs ( Fig. 3(c) ). Confocal imaging revealed membrane labeling by R–Cy5 exclusively on T–Cy3 GUVs ( Fig. 3(d) , SFig. 10 ), confirming high specificity in signal transduction. Quantitative analysis demonstrated that either complete (52 ± 5%) or partial (3 ± 2%) T–Cy3 detected GUVs showed efficient signal transduction via membrane Cy5 labeling. While remaining GUVs exhibited either no T–Cy3 recognition as indicated by filled Cy3 GUVs and no signal transduction (39 ± 5%), or T–Cy3–recognized GUVs showing no R–Cy5 labeling (6 ± 2%, Fig. 3(e) left), indicating a lack of signal transduction. Further, a minimal Cy3 and Cy5 labeling (2 ± 1%) on control GUVs (ATTO488) ruled out the false positives from external quenched targets ( Fig. 3(d–e) right). The minimal signal on control GUVs are attributed to sample handling, GUVs fusion during probe incubation. Optimized membrane fluidity enhances HALOS–mediated signal transduction Membrane fluidity, a critical physical property influenced by lipid compositions, significantly impacts the function of membrane–associated nanodevices, particularly for the insertion and migration of charged DNA species. Using GUVs composed of POPC, a major mammalian membrane component, and the more biologically relevant EggPC, we systematically tuned membrane fluidity by varying core components. 59 , 60 Cholesterol increases membrane order and rigidity, while 18:1 LysoPC (LPC) enhances fluidity and curvature, known to influence ion channel activity and signaling. 59 , 61 All–atom MD simulations revealed that the lipid compositional changes significantly altered the lipid bilayer thickness, head group density, lipid order parameters, and area per lipid, suggesting introduction of LPC increases the fluidity of phospholipid membrane ( SFig. 11 ). Experimentally, Laurdan generalized polarization (GP) calculation 62 and imaging demonstrated a significant alteration of membrane order and fluidity induced by cholesterol and LPC ( SFig. 12 ). 63 Specifically, 30% cholesterol in POPC were highly ordered (GP > 0.4), whereas adding LPC to POPC and EggPC demonstrated highly disordered and fluidic membrane (GP < –0.15). Systematic studies in GUVs reveal that HALOS–mediated signal transduction is strongly dependent on lipid phase state and fluidity. Increasing fluidity promotes lipid disorder, enhancing water penetration and amphiphilic molecular mobility within the bilayer, which facilitates DNA strand hybridization and efficient signal propagation across membrane. 64 The dynamic phase associated with LPC–rich membranes enhances lateral lipid diffusion and reduces the free energy barrier for DNA hybridization, enabling rapid signal transduction. Increasing fluidity, particularly via LPC addition, boosted signal transduction by Cy5 labeling from 31 ± 5% in POPC–only to 66 ± 5% in 30% (mol/mol) (LPC+POPC) GUVs, whereas 42 ± 5% in EggPC to 83 ± 4% in 10% (mol/mol) (LPC+EggPC) GUVs ( Fig. 3(f) , SFig. 12 ). Cholesterol–enriched POPC showed intermediate efficiencies (53 ± 5%). Notably, addition of HALOS 31 (250 nM) did not alter intrinsic membrane order across compositions ( SFig. 12 ), confirming that signal enhancement derives from changes in lipid environment rather than sensor effects. These data establish a direct correlation between increased membrane fluidity, lowered lipid order, and improved HALOS–mediated transduction efficiency. HALOS 31 enables lysis–free detection and transmembrane signaling of long cytosolic nucleic acid targets HALOS 31 achieves exceptional 90 ± 1% efficiency in detecting long nucleic acid targets, establishing robust transmembrane signaling for clinically relevant RNA biomarkers. Long cytosolic RNAs are critical disease biomarkers but remain challenging to detect in live cells without lysis or complex delivery methods. Systematic validation using synthetic 100–nt DNA targets embedded with HALOS 31 recognition domain (41–nt) encapsulated within GUVs demonstrated consistent 97-100% detection (Cy3) and 87-98% signal transduction (Cy5) efficiency across multiple target positions ( Fig. 4(a–c) , SFig. 13 ). This performance remained stable regardless of recognition site location within the long sequence (E1–E3), confirming HALOS’s reliability for detecting diverse DNA targets in GUVs. A quantitative detection and signal amplification by these extended targets is likely due to longer time required for DNA hybridization across membrane resulted in long-lived nanopores on membrane. A comparatively low signal transduction efficiency for E3 targets might be due to ineffective DNA hybridization across membrane using E3 extension (59-nt) from 5 ′ end of T-Cy3 (41-nt). These results establish HALOS 31 as a breakthrough molecular devices for lysis–free detection of longer nucleic acid biomarkers in live cell diagnostics. DNA–RNA hybrids are inherently less stable than DNA–DNA duplexes, posing a thermodynamic challenge for RNA target–triggered hairpin opening. To validate RNA–triggered transmembrane signaling, we encapsulated 2 ′ –O–methyl RNA targets (OMe–T–Cy3, 41 nt) within GUVs and demonstrated successful HALOS 31–mediated detection with 87±4% efficiency ( Fig. 4(d–f) , SFig. 13 ). Signal transduction efficiency of 58 ± 5% confirms HALOS’s robust performance despite the reduced stability of RNA–DNA interactions. This breakthrough enables real–time, lysis–free detection of viral RNA, mRNA biomarkers, and therapeutic targets in living cells, overcoming a fundamental limitation that has restricted RNA–based diagnostics to post–lysis analysis. Membrane–localized HCR achieves 8.2× signal amplification for lysis–free RNA detection in living cells HALOS 31 enables membrane–localized HCR 65 , 66 , establishing the first lysis and transfection–free method for RNA detection with signal amplification in living cells. Using the exposed b* domain of target–recognized HALOS 31 as an HCR initiator, we demonstrated enzyme–free signal amplification directly on live cellular membranes by nodal abstraction and schematic reaction graphs ( Fig. 5(a–b) ). Systematic validation using NUPACK and gel electrophoresis confirmed specific HCR product formation only when both metastable hairpin amplifiers were present (H1, H2), establishing robust target–dependent amplification ( SFig. 14 ). The results showed a higher–order HCR assembly in solution exclusively in the presence of target–detected HALOS 31, with no amplification products detected in negative controls. Membrane–localized HCR achieves 4.2× signal amplification, establishing robust target–dependent enhancement in GUVs. Confocal microscopy revealed Cy5 amplification exclusively in target–containing GUVs, with no signal detected in negative controls lacking either HALOS 31 or T–Cy3 ( Fig. 5(c) , SFig. 15 ). Comparing HCR assembly images to H1 alone revealed a significant increase in membrane Cy5 intensity. Quantitative flow cytometry demonstrated significant signal enhancement when both H1 and H2 monomers were present (3017 ± 1591) compared to H1 alone (1058 ± 491), confirming HCR efficiency on membrane ( Fig. 5(d) ). The large standard deviations reflect heterogeneity in GUV size and target encapsulation efficiency, consistent with single–vesicle measurements. After background subtraction using negative controls without HALOS 31 (450 ± 394), the net amplification was calculated ∼ 4.2× (2,567 vs. 608 a.u.). Three–dimensional imaging confirmed spatial confinement of HCR products on GUV peripheral membrane, establishing membrane–localized amplification architecture ( SFig. 15 ). These results demonstrate that HALOS 31–initiated HCR overcomes the sensitivity limitations of single–molecule detection while maintaining high specificity for intracellular targets. Next, HALOS 31 achieves exceptional ∼ 8.2× signal amplification in live cells, establishing the first lysis–free method for sensitive intracellular RNA detection with HCR signal amplification. While detection of endogenous mRNA requires target–specific sensor redesign and extensive optimization, we validated our method using synthetic 2 ′ –O–methyl RNA targets (2 ′ –OMe–T–Cy3, 100 nM) as proof–of–concept. Lipofectamine–mediated delivery into live A549 and HEK293T cells and HALOS–mediated HCR assembly on cell surfaces, with Cy3 and cy5 labeling demonstrated successful target detection and signal enhancement ( Fig. 5(e) , SFig. 16 ). Comprehensive specificity controls showed no signal in cells lacking HALOS 31, target RNA, or containing scrambled HALOS sequences, establishing high selectivity with minimal background ( Fig. 5(e) , SFig. 16 ). Confocal microscopy revealed HALOS–mediated target detection (Cy3) on membrane and elevated Cy5 signal confirmed robust signal amplification by HCR comparing H1 alone ( Fig. 5(e) , SFig. 16 ). Quantitative analysis revealed 6.5 amplification by confocal microscopy and 8.2× enhancement by flow cytometry (1,739 ± 156 a.u. vs. 360 ± 45 a.u.), confirming consistent amplification across different detection methods ( Fig. 5(f–g) , SFig. 16 ). This strategy overcomes fundamental sensitivity limitations of single–molecule RNA detection while maintaining cellular viability, enabling real–time monitoring of low–abundance intracellular targets. This breakthrough establishes membrane–confined HCR as a powerful tool to study live cell RNA biology, with immediate applications in disease diagnosis, therapeutic monitoring, and fundamental research. Dual–reporter labeling validated complete DNA branch migration across membrane Dual–reporter labeling analysis confirms complete DNA branch migration across lipid bilayers, establishing quantitative evidence for transmembrane target translocation in HALOS–mediated signaling. HALOS 31 insertion and target recognition might induce toroidal nanopore formation, creating a hydrophilic microenvironment within the hydrophobic lipid bilayer, 38 , 45 that enables T–Cy3 to hybridize with HALOS across the bilayer and promote spontaneous HALOS 31 hairpin opening outside the membrane, leading to the formation of thermodynamically stable complexes with target DNA, as supported by molecular dynamics simulations. To validate this hypothesis, we designed an extended target strand (T ′ –Cy3) with an extended branch migration domain for orthogonal reporter labeling (R–AF488). Successful transmembrane migration would yield both Cy5 and AF488-rings on GUVs by labeling the open hairpin (b*) and extended target (d) domains. NUPACK analysis and gel electrophoresis established reporter orthogonality with no cross–reactivity, while solution–phase dual–labeling validated the experimental design ( SFig. 17 ). Individual reporter labeling of T ′ –Cy3–detected HALOS on GUVs established specificity, with confocal imaging showing no inter–channel fluorescence bleed–through ( Fig. 6(c) left and middle, SFig. 18 ). Subsequent incubation with both reporters produced simultaneous dual labeling, confirming complete T ′ –Cy3 hybridization with HALOS stem across the lipid membrane during target detection and hairpin opening( Fig. 6(c) right, SFig. 17 ). Experiments with live HEK293T cells validated cellular compatibility, with co–localization of both reporters on cell membrane following external HALOS 31 anchoring and T ′ –Cy3 targeting ( SFig. 18 ). Quantitative analysis of dual reporter–labeled GUVs from the T ′ –Cy3 detected pool revealed 53 ± 2% labeling with R–Cy5 and 38 ± 4% with R–AF488 (n=200 GUVs each) ( Fig. 6(d) ). The elevated frequencies of R-Cy5 labeling revealed that hairpin opening process surpass the extended target DNA branch migration events across membrane. The slightly lower AF488 labeling likely reflects incomplete T ′ –Cy3 migration events within the experimental time frame (120 min). These results establish quantitative evidence for transmembrane DNA branch migration in the HALOS system. Proximal split–initiator induced HCR ( ψ –HCR) validates complete transmembrane branch migration with 5× signal amplification Proximity–dependent ψ –HCR design achieves 5× signal amplification while confirming complete transmembrane DNA branch migration through proximity–induced initiator formation and activation of HCR. This innovative approach combines sequences from the extended target (T ′ ) and open hairpin to create split–initiator domains that enable ψ –HCR initiation only when both are present in close proximity, thereby minimizing non–specific background signal. Absence of either initiator prevents amplification, as validated by gel electrophoresis showing signal amplification exclusively when both split initiators are present in solution ( SFig. 19 ). We then applied this principle to T ′ –Cy3 branch migration on HALOS 31, performing membrane–localized ψ –HCR ( Fig. 6(e–f) ). GUV–based validation established proximity–dependent amplification following transmembrane branch migration. Confocal microscopy revealed enhanced Cy5 signal intensity compared to H1 labeling alone, establishing efficient ψ –HCR amplification on membrane ( SFig. 20 ). Control images with single initiator domains (T–Cy3) eliminated HCR propagation, demonstrating minimal false–positive amplification and validating the proximity requirement of initiator domains. Signal amplifications in A549 cells demonstrated robust ψ –HCR performance with quantitative signal enhancement. Confocal microscopy showed successful 2 ′ OMe–T ′ –Cy3 detection with HALOS 31 anchored on cell membranes (Cy3) and distinguished signal amplification (Cy5) compared to H1 reporter alone ( Fig. 6(g) , SFig. 12 ). Control experiments lacking split-initiator domain produced only Cy3 labeling, validating specificity. Quantitative analysis revealed ∼ 5× signal enhancement by confocal microscopy ( Fig. 6(h) ) and ∼ 2.6× amplification by flow cytometry ( Fig. 6(i) , SFig. 12 ), with differences reflecting detection sensitivity variations. These results demonstrate that proximal split initiator formation on HALOS mediated signaling and ψ -HCR enables specific transmembrane signal amplification with quantitative enhancement and minimal background interference. Discussion Here, we present a transmembrane protein mimic using an amphiphilic DNA hairpin, which showed ability to detect intra-vesicular and cellular nucleic acid targets without altering the membrane integrity. By covalent anchoring only two cholesterol into internal HALOS domain and without any charge neutralization coupling to DNA phosphate backbone, we introduce a transmembrane domain in HALOS 31 to withhold the nanosensor within the hydrophobic bilayer of lipid membrane. 45 , 53 , 58 MD simulations reveal a robust incorporation and extended stability of several variants and conformations of HALOS within membrane. Two cholesterols with ∼ 180 ° angle between them makes the axial orientation of HALOS alone and target detected HALOS within membrane with preferred conformation. Live-cell microscopy showed that even after repetitive washing and dilution cycles, HALOS 31 remains strongly adhesive to the membrane and display membrane insertion properties via detecting intracellular targets. Our studies revealed that target detection followed by conformational switch of HALOS 31 across membrane leading signal transduction by reporter labeling. Transmembrane proteins, especially GPCRs transduce signals ‘outside-in’ across membrane by ligand binding outside, rapid conformation changes in G-protein monomers and GDP-GTP conversion followed by downstream intracellular signaling cascades. 7 , 67 Various isoforms of GPCRs involved in integrins ‘inside-out’ signaling display the unique directional properties of cell adhesion, spreading and mechanotransduction. 68 – 70 In this study, we showcase a synthetic mimic of GPCRs which transduce signal ‘inside-out’ and vice versa based on the direction of ligand-target addition. Since till date, ∼ 35% of all FDA approved drugs are targeted to GPCRs 4 , 10 , 71 , 72 , mimicking their function using HALOS 31 remains of great interest for therapeutic activation, intracellular signaling cascade generation. The current HALOS-mediated nucleic acid detection system utilizes synthetic target sequences limited in length upto ∼ 100-nt due to synthesis constraints, without considering secondary structures typical of cytosolic short non coding RNAs such as miRNAs or tRNAs. Detection of longer cytosolic RNAs like mRNAs, which possess highly complex secondary structures, was not pursued in this study. At present, each HALOS variant is capable of detecting only a single target sequence, with multiplexed detection yet to be explored in future studies. Temporal limitations of detection and signal amplification, on the scale of minutes to hours, are mainly attributed to the slow DNA branch migration across the membrane and the kinetics of in situ HCR on live cell membrane. Enhancing multiplexed detection and temporal resolution will necessitate the development of HALOS variants that circumvent the requirement for DNA strand translocation across membranes and incorporate faster DNA-based isothermal amplification strategies to boost signal output and reduce response times. 73 Besides, incorporation of 18:1 LPC, a monoacyl phospholipid, into biologically relevant membranes (POPC and EggPC) significantly influences membrane fluidity, enhancing HALOS-mediated signal transduction events. LPC induces positive membrane curvature and disrupts lipid packing 74 , with bilayer integrity maintained up to approximately 30% LPC (molar ratio) in POPC and only about 10% in EggPC membranes, beyond which bilayer formation is compromised. This disruption reflects greater disorder in EggPC compared to POPC bilayers. Modulation of membrane biophysical properties by LPC incorporation improves liposome-based therapeutic delivery by enhancing membrane permeability and faster delivery approach. Moreover, LPC can alter cell membrane permeability to the extent of inducing necrotic cell death, highlighting its physiological and pathological significance in cell signaling and membrane dynamics. Also, studies have shown that LPC can alter the cell membrane permeability, which can lead to necrotic cell death, thus deciphering physiological and pathological significance. 75 Thus, LPC incorporation provides a strategic avenue to tune membrane characteristics for improved biointerfacing and therapeutic outcomes in membrane-based synthetic biology applications. The sequence-specificity in HALOS-mediated signal transduction system from a pool of synthetic cells mixture demonstrates high molecular specificity by triggering inside-out signaling exclusively in cells containing a defined target nucleic acid sequence, while cells without this sequence remain inactive. This precise target recognition enables discrimination within heterogeneous cell populations, thereby facilitating efficient enrichment via flow cytometry-based isolation without affecting traditional cell surface receptor based isolation strategies and preserving their biological functions. Such specific activation and sorting are critical for cell-based therapies; for instance, engineered T–cells recognizing cancer-specific nucleic acid markers can be selectively isolated for adoptive cell therapies targeting tumors or hematologic malignancies. 76 Similarly, stem cell-based immunotherapies benefit from isolating populations modulated by defined molecular signals. 77 Thus, HALOS-mediated signaling constitutes a programmable platform for sequence-specific cell recognition and purification, potentially enhancing the purity, efficacy, and safety of cell-based therapeutics in oncology. This approach builds on the paradigm of artificial transmembrane signaling in synthetic cells, where membrane-anchored synthetic receptors mediate targeted, non-diffusive transmembrane signal transduction analogous to natural receptor mechanisms. Finally, sensitive, lysis-free detection of intracellular targets coupled with HCR-based signal amplification offers substantial biological and diagnostic advantages. In situ HCR, leveraging programmable DNA hairpins, enables enzyme-free, isothermal, highly specific amplification under physiological conditions, preserving cell viability and avoiding genetic manipulation. 78 , 79 Advances in branched HCR designs further enhance efficiency, sensitivity and specificity in fixed cell mRNA imaging. 80 . HALOS integrates membrane-anchored DNA nanodevices with HCR, enabling amplified detection of low-abundance RNAs in live cells, avoiding complex intracellular delivery or genetic engineering, facilitating applications such as real-time imaging of gene expression, extracellular mRNA biomarkers, at the single-cell level. Compared to molecular beacons 81 , FISH 82 , and genetically encoded sensors 83 , which require delivery, cell fixation, or genetic manipulation and often lack amplification, HALOS-HCR offers amplified signal transduction on live membranes with minimal perturbation. Looking forward, combining lysis-free HALOS-HCR signal amplification platform on live cell membranes promises early biomarker detection, therapeutic monitoring, precise cell isolation and advances in cell-based therapies, bridging synthetic biology, to molecular diagnostics, and translational medicine. Funding The research was funded by the National Institutes of Health (NIH) (1DP2AI144247 to R. F. Hariadi; 1R61CA278558 to H. Yan and R. F. Hariadi) and Flinn Foundation (24-17692) to R.F. Hariadi. SERB startup research grant (SRG/2022/002109) and the DST Inspire faculty fellowship, India (IFA20-PH-256) to H.J. G.B.M. Wisna was supported by an American Heart Association (AHA) predoctoral fellowship (23PRE1029870). Conflicts of Interest RFH and HY are among the scientific co-founders of Exodigm Biosciences and hold an equity interest in the company. Their interests were reviewed and managed in accordance with his institutional conflict-of-interest policies. Correspondence All correspondence and requests for materials should be addressed to R. Sasmal and R.F. Hariadi. Methods DNA-cholesterol synthesis using DNA synthesizer Cholesterol-conjugated DNA was either obtained from Integrated DNA Technology or synthesized in the laboratory using a DNA synthesizer (Applied Biosystems 3400 model). The synthesized DNA was cleaved from the solid CPG support using a 30% ammonia solution, in accordance with the manufacturer’s protocol. The crude DNA were then purified using Agilent 1260 HPLC and analyzed using Agilent 6530 Quadrupole Time-of-Flight ESI-MS. The pure DNA-cholesterol conjugates were then reconstituted in ultrapure water and stored at –20 °C in lyophilized form of 10 µM x 20 µL aliquots. DNA-fluorophore conjugation Both 3 ′ and 5 ′ modified DNA-fluorophores were either synthesized using DNA synthesizer or by amine-NHS ester coupling methods. Cy3 and Cy5 fluor conjugated DNA were synthesized using DNA synthesizer while AF488 fluor coupling with DNA was carried out using amine modified DNA and AF488 NHS ester (Lumiprobe, 11820). In a typical conjugation method, aqueous solution of amine-modified DNA in 0.1 M sodium bicarbonate buffer was mixed with AF488 NHS ester (5 equiv.) in 10% DMSO (v/v) for 12 hours. The conjugates were then purified by HPLC and analyzed using ESI-MS. Molecular dynamics simulations All-atom MD simulations were performed using NAMD3 84 with periodic boundary conditions and the particle mesh Ewald (PME) method implemented for long-range electrostatics 85 . The simulations were conducted in the isothermal-isobaric ensemble, with pressure and temperature regulated using the Nosé–Hoover Langevin piston 86 , 87 and Langevin thermostat 88 respectively. For membrane systems, anisotropic pressure coupling was applied to maintain a constant ratio between the X and Y dimensions, allowing the Z dimension (normal to the membrane) to fluctuate independently. The CHARMM36 force field 89 were used describe all bonded and non-bonded interactions among DNA, lipid bilayer membranes, water, and ions. Parameters for cholesterol and the TEG linker were adopted from previous studies 45 , 53 , 90 . A cutoff scheme of 8–10–12 Å was employed for van der Waals and short-range electrostatic interactions. All systems were simulated using a 2 fs time step for integrating the equations of motion. The SETTLE algorithm 91 constrained water geometry, and all other covalent bonds involving hydrogen were constrained using the RATTLE algorithm 92 . Coordinates were saved every 19.2 ps. Simulation trajectories were analyzed and post-processed using VMD 93 and CPPTRAJ 94 . Initial PDB structure of DNA hairpins were created with Alphafold3 server 95 , yielding plausible models with a pTM-score of 0.4. An additional phosphate group was incorporated at the DNA backbone to enable covalent attachment of the cholesterol-TEG linker using the psfgen plugin in VMD 96 . In-house TCL scripts were used to construct models with DNA hairpins anchored in phospholipid membranes, positioning cholesterol-anchored HALOS in both axial and lateral orientations relative to POPC bilayers. All membrane lipids within 0.1 Å of DNA hairpin or cholesterol atoms were removed, and the number of lipids was balanced between upper and lower leaflets. All the systems were solvated with TIP3P water box. Potassium and chloride ions were added to a 0.15 M concentration of KCl in the system using the Autoionize plugin of VMD. Thus assembled all atom models of all the simulated systems are summarized in Supplementary table 6 and 7. Following assembly, each system underwent 4,800 steps of energy minimization with the conjugate gradient method. The minimized system was equilibrated in the NPT ensemble (P = 1 bar, T = 300 K) for 50 ns with harmonic restraints (1 kcal mol − 1 Å − 2 ) applied to all non-hydrogen atoms of the DNA channels, referenced to their initial coordinates. For membrane-embedded and aqueous systems, restraints were maintained for 50 ns and 5 ns respectively; followed by ∼ 1,000 ns of unrestrained equilibration. Hydrogen bond and cholesterol angle analysis Hydrogen bond analysis was carried out using a custom TCL script. A hydrogen bond was defined by a donor–acceptor distance cutoff of 3 Åand a donor–hydrogen–acceptor angle ≤ 45 ° . We computed Watson–Crick (WC) hydrogen bonds between paired DNA bases for each frame of the simulation. For visualization, the mean hydrogen–bonds per frame was calculated per frame of simulation (1 ns each) and plotted as gaussian distribution plots. For Broken base pair representation, the hydrogen bond counts were normalized by the total number of base pairs in each system and represented as heatmaps. The cholesterol angles were determined by considering all atoms from cholesterol molecules and generated a vector considering DNA stem within membrane as perpendicular axis. The angle between two cholesterol vectors were measured per frame of simulation time (1 ns each) and plotted in circular plot ranging 0-360 ° angle. Gel electrophoresis The native polyacrylamide Gel electrophoresis (PAGE) with 8% (w/v) precast gel in 1x TBE + 12.5 mM MgCl 2 was performed for the signal transduction and HCR using 1x TBE buffer (89 mM Tris+89 mM Boric acid+2.0 mM EDTA, pH 8.4) supplemented with 12.5 mM MgCl 2 . The DNA strands were annealed using standard protocol. Samples were heated to 90 °C, gradually cooled up to 76 °C at the rate of 2 °C/5 min and then slowly cooled down to 24 °C at the rate of 4 °C/5 min followed by stored at 4 °C. The DNA were loaded into gel with a typical concentration of 10 pmol (1 µM x 10 µL) and run at 100 V for 60 min. The gel was then post stained with 0.5 µg/mL ethidium bromide (EtBr) solution for 10 min and viewed under UV transilluminator. 1% (w/v) Agarose gel electrophoresis was carried out for HCR using 1x Sodium Borate buffer (10 mM NaOH, pH adjusted to 8.5 with boric acid) supplemented with 12.5 mM MgCl 2 . The gel was prestained with SYBR gold (0.5 µg/mL), loaded with 10 pmol (1 µM x 10 µL) sample and run with 100 V for 90 min under ice cold condition and imaged using UV transilluminator. GUV preparation Cy3 conjugated targets (T-Cy3) encapsulated GUVs were prepared using standard electroformation protocol. 57 Briefly, in this study, 10 mM lipid stocks (∼ 5 µL) in chloroform (Avanti polar lipids: POPC, 850457; EggPC, 840041; 18:1 LPC, 845875; purity> 99%) were homogeneously spread over the conductive side of the indium tin oxide (ITO) coated two glass slides and evaporated the solvent under vacuum for 20 min. All these lipid stocks were stored in chloroform at −20°C for long time (1-2 years) storage and allowed to warm them up at room temperature before every use. For the preparation of GUVs with mixed lipids, the lipid stocks were premixed in molar ratio within a glass vial prior deposition to ITO glass slides. A rubber ring was then placed on the lipid coated ITO slide and filled with 250 µL of 300 mM sucrose solution (∼ 300 mOsm/kg) containing 250 nM T-Cy3. Another ITO coated glass was placed on top, and the chamber connected to the electrode of the Vesicle Prep Pro (Nanion). An AC field (3 V, 10 Hz) was applied via the electrodes for 128 min while the solution was heated to 37 °C. The prepared GUVs were collected immediately after the electroformation and stored at 4 °C and used up to a week. Confocal microscopy The confocal images were captured using Nikon AX R Ti2 laser scanning confocal microscope using a plan apochromat 60x oil OFN25 DIC N2 objective (NA 1.42). Four monochromatic lasers (405, 488, 561 and 640 nM) have been used for the excitation of the respective fluorophores. The emissions were filtered using respective highly sensitive GaAsP adjustable emission filters (503-530 nM for 488 laser line, 571-625 nM for 561 nM laser line and 662-737 nM for 640 laser line respectively). The laser intensity and detector gain were kept constant for each channel while confocal scanning. The multichannel images were recorded using sequential scanning with 1024 x 1024 pixel resolution using 2x averaging and 1 AU pinhole size. 3D confocal images were recorded using 512 x 512 pixel in XY and 500 nm in Z-direction resolution. A total 31 number of slices were recorded over 15 µm depth of GUVs sample and 3D images were reconstructed using ImageJ 3D viewer plugin. Membrane fluidity measurement The membrane fluidity of each lipid compositions within GUVs was measured using a membrane sensitive probe, Laurdan (MedChem Express, HY-D0080) and calculated their GP values by the following generalized polarization equation: GP= [I B -I R ]/[I B +I R ]. Laurdan (5 µM) was incubated with freshly prepared GUVs in 300 mM sucrose solution for 15 min and subjected to plate reader for fluorescence measurement across the wavelength range (430-600 nM with 1 nM steps) after excitation at 390 nM (Laurdan). The GP values were calculated considering I B at 436 nM and I R at 512 nM. For fluorescence spectral scanning imaging using confocal microscopy, the GUVs incubated with Laurdan were excited with 405 nm laser and spectral scanning was carried out using a narrow 5 nM emission window (420-600 nM). The GP images were generated using I B and I R confocal spectral scanning images with the help of custom built GP plugin in ImageJ. Sample preparation for signal transduction across GUVs membrane Electroformed GUVs(∼100 µL) were centrifuged at 300 g for 5 min after dilution with 1 mL homo-osmotic (∼300 mOsm/kg) 1x imaging buffer (1x TAE+150 mM NaCl+12.5 mM MgCl 2 ) and exchanged the outside buffer to remove the excess outside T-Cy3. Post centrifugation, the supernatant was discarded and the GUVs pallets were further suspended with 100 µL fresh 300 mM sucrose solution and can be stored at 4 °C for at least 7 days. Excess of complementary DNA (5 µM, 20x excess) against T-Cy3 (250 nM) was added to GUVs solution for 1 h to absolutely quench the T-Cy3 traces outside the GUVs. For experiments with mixed GUVs, the GUVs were mixed thoroughly prior to complementary addition. Then, annealed HALOS 31 (250 nM) was incubated with ∼1-2 µL GUVs in 100 µL 1x imaging buffer within a coverslip bottom 96 well plates (Ibidi, 89621) for sufficient times (2-3 h) for the sensor insertion and TMSD across the GUV membrane. R-Cy5 (100 nM) was then incubated with the GUVs for 60 min and then diluted to ∼500 µL using 1x imaging buffer to reduce the background signal of R-Cy5 prior proceeding to confocal microscopy. RNA transfection protocol for live-cell imaging and flow cytometry HEK293T and A549 cells were plated at 70–90% confluency either in 8-well glass-bottom chamber slides (Ibidi, 80806) for confocal microscopy or in 6-well dishes (Greiner bio-one, 657160) for flow cytometry. For healthy populations of cells Prior to transfection, culture media was replaced with Opti-MEM (200 µL for confocal; 2 mL for flow cytometry) and incubated for 1 hour. For confocal imaging, 2 ′ -OMe RNA target (100 nM) was mixed with 1.5 µL P3000 reagent and diluted in 50 µL Opti-MEM, while Lipofectamine (1 µL) was diluted separately in 50 µL Opti-MEM; both solutions were vortexed, centrifuged, combined by pipetting, and incubated for 15 min before drop wise addition to cells. For flow cytometry, 100 nM target was mixed with 7.5 µL P3000 reagent diluted in 75 µL Opti-MEM, and 5 µL Lipofectamine was diluted in 75 µL Opti-MEM; similarly mixed and incubated before addition. Transfections were conducted under standard culture conditions. After 60–90 min, cells were washed three times with DPBS (1 min incubation each) to remove extracellular non-transfected RNA and processed for imaging or flow cytometry. HCR protocol HCR were performed in solution using 1x imaging buffer for characterization using gel electrophoresis. 3 µL each stock of 1 µM H1, H2 and initiator (HALOS 31+T-Cy3) were mixed in a PCR tube for the HCR. In the absence of any reactant, 3 µL of 1x imaging buffer was added to adjust the total volume of 9 µL in sample mixture. For completeness, the reaction were allowed at room temperature for 24 hours before characterization using gel electrophoresis. To perform HCR on GUVs surface, HALOS 31 (2.5 µM x 10 µL) was incubated with T-Cy3 encapsulated GUVs solution in 100 µL 1x imaging buffer for 3 hours to perform TMSD across membrane followed by addition of H1 and H2 (1 µM x 10 µL each) to the GUVs solution. After 1 hour, confocal microscopy images were acquired after dilution to ∼500 µL using 1x imaging buffer. For HCR on live cell membrane, 2 ′ -OMe-RNA–Cy3 (2 ′ -OMe–T-Cy3, 41-nt) transfected cells in 8-well coverslip bottom chamber slides (Ibidi, 80806) were subjected to remove the extracellular targets by repetitive washing using DPBS (3 times), and treating with complementary DNA (4 µM, 40x excess) in opti-MEM media supplemented with Aurintricarboxylic acid (ATA, a nuclease inhibitor; 50 µM) and MgCl 2 (12.5 mM) for 20 min in live cell culture conditions for complete quenching of extracellular non-transfected RNA. The cells were then washed with DPBS (3 times) and treated with HALOS 31 (250 nM) in Opti-MEM media supplemented with ATA (50 µM), MgCl 2 (12.5 mM), and complementary DNA (2 µM) for 20 min at 37 °C. H1 and H2 (250 nM each) diluted in opti-MEM (200 µL), supplemented with 50 µM ATA and 12.5 mM MgCl 2 were then incubated with the cells for 30-60 min at 37 °C. Similarly, ψ -HCR were performed by transfecting 2 ′ -OMe-T ′ -Cy3 (60-nt) and choosing respective H1 and H2 reporters. 2 ′ -OMe–T-Cy3 (41-nt), lacking the extended split-initiator domain was considered as a negative control for ψ -HCR. The cells were finally washed with DPBS, exchanged the opti-MEM media and proceed for confocal microscopy. Flow cytometry 2 ′ -OMe RNA transfected cells from 6 well-dish were washed, trypsinized, centrifuged at 800 rpm for 4 min and resuspended using opti-MEM media in a microcentrifuge tube. Complementary DNA (4 µM, 40x excess) was treated to cells for 20 min at 4 °C. Cells were then washed out by centrifugation and 200 µL HALOS 31 (250 nM) was incubated for 20 min at 4 °C. After excess unbound sensor removal by centrifugation, the reporters (H1 and H2, 250 nM each) diluted in opti-MEM (200 µL) were then incubated with the cells for 30-60 min at 4 °C. The cells were finally centrifuged, removed excess unreacted reporters and diluted using opti-MEM media and proceed for flow-cytometry. Similarly, ψ -HCR were performed by transfecting OMe-T2-Cy3 (60-nt) and choosing respective H1 and H2 reporters. OMe-T1-Cy3 (41-nt) was considered as a negative control for ψ -HCR. The individually prepared samples were finally diluted to 500 µL using opti-MEM media and subjected to flow cytometry analysis. 10,000 scattering events were collected upon excitation of Cy3 (561 nM) and Cy5 (642 nM) dyes and the live cells were adequately gated from the scattered plot of single cells. Image analysis The confocal images were post-processed using ImageJ for representation. Each channel images were kept with the same intensity window for comparison. The GP images were generated using a custom-coded ImageJ macro plugin. High-throughput analysis on GUVs were performed by isolating each GUVs coordinates using DisGUVery 97 and performing algorithm based membrane intensity calculation using custom coded Mathematica programming. The intensity plots and standard deviations were generated using GraphPad Prism and Mathematica. High-throughput image analysis of live cells were performed by analyzing kymograph on the cell membrane using custom-coded ImageJ macro plugin and Mathematica programming. Quantification and data analysis All graphs were created using GraphPad Prism 9 or Mathematica coding. Error bars represent S.d. unless otherwise noted. For comparison between two groups, P values were determined using two-tailed Student’s t-tests. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; NS, not significant. The bootstrap method was used to calculate error using standard deviation (s.d.) of the mean of Cy3 and Cy5 ring counts for GUVs. The bootstrap calculation was performed using a custom MATLAB code. First, the Cy3 and Cy5 rings were assigned as binary numbers 0 and 1 for negative or positive membrane labeling, respectively. For bootstrapping, N = 1,000 simulated data were generated by random resampling with replacement from the original dataset, preserving its size in each iteration. Each resampled dataset was stored as an independent bootstrap realization, from which the mean value was computed iteratively to obtain the bootstrap distribution of the mean. Based on this distribution, the final mean value and s.d. were determined across all bootstrap realizations of the Cy3 and Cy5 ring content, thereby quantifying the uncertainty of the estimated mean without assuming an underlying parametric distribution. Code Availability The scripts and code for processing the data can be found in https://github.com/rhariadi/artificial-gpcr Supplementary Information Supplementary Materials and Methods Unmodified DNA were purchased from Integrated DNA technologies (IDT). Modified DNA were synthesized in the laboratory using a solid-phase DNA synthesizer (Applied Biosystems 3400). The reagents and phosphoramidites were purchased from Glen Research, Sigma Aldrich, and Lumiprobe. Synthesized DNA were purified by high-performance liquid chromatography (HPLC, Agilent 1260) using a c18 reverse phase column. The solvents used as mobile phase in HPLC were solvent A: 100 mM Triethylammonium Acetate buffer, pH 7.0 (TEAA; Sigma Aldrich, 625718) and solvent B: Acetonitrile (Sigma Aldrich, 439134). The DNA conjugates were analyzed by mass spectrometry using electrospray ionization (ESI-MS) method equipped with Agilent 6530 Quadrupole Time-of-Flight LC/MS System. The intact mass (M) analysis of DNA was carried out by performing deconvolution using BioConfirm software (Agilent). Unmodified DNA purchased from IDT were filtered through molecular weight cut off (MWCO) filter to remove all traces of salts and reagents using ultrapure water. The stocks of all DNA were adjusted to 100 µM in ultrapure water prior to the experiments. Molar extinction coefficient ( ε ) of all DNA were obtained from IDT DNA analysis website. The Native PAGE analysis was performed using Bio-Rad Mini-PROTEAN Tetra Vertical Electrophoresis Cell (Bio-Rad, 1658004) and agarose gel electrophoresis was performed using Thermo Fisher Scientific Owl Easy Cast (B2-BP) setup. The GUVs formation were carried out using a Nan]i[on vesicle prep pro setup (Nanion technologies GmbH). Confocal images were acquired using a Nikon AX R confocal setup equipped with a GaAsP detector, and image analysis were carried out using Fiji-ImageJ, DisGUVery, custom-coded Mathematica and MATLAB. In-house DNA synthesis, conjugation, and characterization HALOS 14 and HALOS 31 were purchased from IDT, while HALOS 21–28 were synthesized in-house using an automated DNA synthesizer via standard solid-phase phosphoramidite chemistry in the 3 ′ to 5 ′ direction. The synthesis cycle included sequential detritylation, coupling of phosphoramidite monomers, capping. and oxidation steps to ensure high coupling efficiency. Following chain assembly, oligonucleotides were cleaved from the solid support, deprotected, and purified using reverse-phase HPLC. Fluorophores (Cy3, Cy5, ATTO488), and biotin modifications were introduced either during synthesis via pre-labeled phosphoramidites or post-synthetically using NHS-ester–amine conjugation chemistry. Purified conjugates were further analyzed by ESI-MS equipped with BioConfirm to confirm intact molecular masses. For ESI-MS, ∼10 µM DNA samples (10 µL) were injected in ultrapure water, using 0.1% NH 4 OH in ultrapure water as mobile phase. Modeling membranes with varied lipid compositions All simulated membrane systems contained 64 lipid molecules per leaflet. Initial bilayer configurations were built using the CHARMM-GUI membrane builder 93 with the following compositions: POPC (64 POPC per leaflet), EggPC (40 POPC and 24 SLPC per leaflet), and POPC:cholesterol (45 POPC and 19 cholesterol per leaflet). Parameters for LysoPC (18:1), not available in CHARMM36, were assembled by extracting the molecular structure from the Human Metabolome Database (HMDB ID: HMDB0010385) and generating parameters using CGENFF 98 . Reference LysoPC species (14:0 and 16:0) in CGENFF provided parameter and charge penalties of 1 and 0.491, respectively, supporting accurate simulation of LysoPC (18:1) in the present study. Structure building and optimization of Cy3 dye The initial structure of Cy3 dimethyl was obtained from PubChem (CID: 5705412) and subsequently modified by addition of ethanol and ethane groups using Gaussian 16. The modified structure underwent geometry optimization and vibrational frequency analysis at the density functional theory (DFT) level, employing the B3LYP functional with the 6-31+G basis set to balance computational efficiency and polarization accuracy. Vibrational frequency calculations confirmed the optimized geometry as a true minimum by the absence of imaginary frequencies. The Gaussian input included iop(6/33=2,6/42=6) keywords to enable detailed natural bond orbital analysis and ensure proper charge distribution for subsequent simulations. Force field parameters for Cy3 were derived using the CHARMM General Force Field (CGenFF) 98 . RMSF and contour length calculation All RMSF analyses were performed over the entire simulation trajectory of 1 µs using CPPTRAJ. The calculations were carried out by selecting the backbone atoms of DNA bases to assess the structural fluctuations. Contour length distributions were obtained by summing the Rise base pair parameter of all paired DNA bases. The Rise values were extracted using the ‘nastruct’ module of CPPTRAJ, and the resulting distributions were used to evaluate DNA overstretching behavior around hydrophobic modifications. Sample preparation of HALOS stability studies in GUVs ATTO488 dyes encapsulated (without target DNA) GUVs were prepared by electroformation and resuspended in imaging buffer (1x TAE+150 mM NaCl+12.5 mM MgCl 2 ). HALOS variants (250 nM) were thoroughly mixed with GUVs and incubated for 30 min at room temperature. Reporter R-Cy5 (13-nt ssDNA, 100 nM) was added and the mixture incubated for 30 min at room temperature. GUVs solution were diluted to ∼ 5x volume using imaging buffer and subjected to confocal microscopy. Cell culture HEK293T and A549 cells were cultured at 37 °C in a humidified atmosphere containing 5% CO 2 using Dulbecco’s Modified Eagle’s Mem (DMEM, high glucose) supplemented with 10% fetal bovine serum (FBS), 1% antibiotics (100 U/mL penicillin, 100 µg/mL streptomycin; Corning, 30-002-CI) and 1% MEM non-essential amino acids (Corning, 25-025-CI). At ∼80% confluence, the cells were washed using DPBS (pH 7.3), trypsinized, and suspended in a culture medium. For imaging experiments, cells were counted and seeded at a density of ∼10,000 cells per well in 200 µL culture media into 8-well glass bottom plates (ibidi, 80806). Cells were incubated under the same conditions for 24 hours to reach 80% confluence before imaging. Sample preparation of HALOS stability studies in live cells HEK293T cells seeded on glass bottom chambers were washed with DPBS, incubated with HALOS variants (250 nM) in Opti-MEM media containing ATA (50 µM) and MgCl 2 (12.5 mM) for 15 min under live cell culture conditions. Unbound probes were removed using DPBS washes (3 times) with 1 min incubation each time. Reporter R-Cy5 (13-nt ssDNA, 100 nM) was incubated with the cells for 15 min in Opti-MEM containing ATA (50 µM) and MgCl 2 (12.5 mM) for 15 min under live-cell culture conditions. Unbound probes were removed using DPBS washes (3 times) with 1 min incubation each time, and cells were maintained in Opti-MEM prior to confocal microscopic imaging. Stepwise sample preparation for signal transduction experiment in GUVs 100 µL T-Cy3 (or ATTO488 dye as control) encapsulated (250 nm) GUVs prepared using electroformation, were diluted with 900 µL homo-osmotic (∼300 mOsm/kg) 1x imaging buffer (1x TAE+150 mM NaCl+12.5 mM MgCl 2 ) in a microcentrifuge tube and centrifuged at 300g for 5 min at room temperature. GUVs settles down as a pallet-like residue and the supernatant was exchanged carefully with 1 ml of fresh 1x imaging buffer. After two repetitive washings, GUVs were resuspended in 100 µL homo-osmotic sucrose solution. The GUVs solution can be stored at 4 °C for at least 7 days. The complementary DNA (5 µM, 20x excess) in homo-osmotic 1x imaging buffer (1x TAE+150 mM NaCl+12.5 mM MgCl 2 ) was added to GUVs and incubated for 60 min at room temperature for complete quenching of extravesicular targets. Annealed HALOS 31 (250 nM) was incubated with GUVs (∼1-2 µL) in 100 µL 1x imaging buffer within a coverslip bottom 96-well plates for 2-3 h at room temperature. R-Cy5 (100 nM) was added and incubated with the GUVs for 60 min. The GUVs suspension was diluted to ∼500 µL with 1x imaging buffer to reduce the background signal of R-Cy5 prior proceeding to confocal microscopy. Calculation of the radius of gyration of 13-nt ssDNA R-Cy5 in water The radius of gyration (R g ) of 13-nt ssDNA R-Cy5 was estimated using a worm-like chain (WLC) model approximation. For a semi flexible polymer of contour length L and persistence length l p , the mean-square radius of gyration is given by The contour length L was calculated from the number of nucleotides ( N ) and the rise per nucleotide ( a = 0.59 nm) for ssDNA in aqueous buffer: The persistence length of ssDNA under physiological ionic strength was assumed to be l p = 1.1 nm, consistent with previous single-molecule studies. 99 Substituting these values into Eq. 1 gives Thus, the radius of gyration of the 13-nt ssDNA–Cy5 conjugate in water was estimated as This value represents the equilibrium spatial extension of the ssDNA–Cy5 conjugate in aqueous solution and is consistent with previous experimental and computational reports for short ssDNA oligomers. Hairpin amplifier stands (H1, H2) design for ψ -HCR Metastable DNA hairpin monomers (18 bp stem/6-nt loop) for ψ -HCR were designed using NUPACK simulations to optimize their thermodynamic and kinetic properties. Initially, the H1 strand was designed to utilize a toehold domain (9-nt) that initiates binding of the first split-initiator (SI-1) from extended target domain (d), followed by subsequent binding of a SI-2 from open hairpin through recognition of a proximal split-toehold, which would open the H1 hairpin stem and trigger HCR propagation from the opened state. However, gel electrophoresis analysis revealed that, although the SI-1 and SI-2 bind the H1-hairpin, it failed to induce efficient stem opening, leading to minimal or no in situ hybridization. This limited propagation was attributed to insufficient thermodynamic stabilization upon binding of the second split initiator. To enhance the HCR propagation, we designed a second class of H1-hairpin by distributing the initiator binding sites differently: the SI-1 from extended target domain (d) binds both the toehold (9-nt) and a partial stem region (8-bp) of H1, while the SI-2 from open hairpin (b*) binds the remaining stem segment (10-bp). This split-stem binding design increases overall binding affinity and significantly improves the thermodynamic stability of the opened H1 conformation. Consequently, this configuration facilitated more robust and faster ψ -HCR polymerization, as confirmed by pronounced polymerization bands in gel electrophoresis ( SFig. 19 ). Sample preparation for live-cell confocal microscopy Live HEK293T and A549 cells were plated at 70-90% confluency into 8-well glass bottom dish using DMEM media. The media was exchanged to opti-MEM prior to 1 h of RNA transfection process. Cells were allowed to remain healthy prior transfection. For each well, Cy3-labeled 2 ′ -OMe modified RNA targets (100 nM) were premixed with 1 µL of P3000 reagent in a microcentrifuge tube and diluted with 50 µL opti-MEM media following mixed well. 1 µL Lipofectamine reagent was diluted with 50 µL opti-MEM media following mixed well. Both of these tubes were vortexed and centrifuged and combined into a single tube by vigorous mixing and allowed to stand for 15 min at room temperature. The cocktail mixture was added drop wise to the live cells and allowed for 60-90 min for transfection process. The cells were then washed thoroughly (3 times) using DPBS to remove the excess non-transfected targets. Complementary DNA (4 µM, 40x excess) against RNA targets supplemented with ATA (50 µM) and MgCl 2 (12.5 mM) was incubated with the cells for 20 min in live-cell culture conditions for complete quenching of extracellular non-transfected RNA. The cells were then washed with DPBS (3 times) and treated with HALOS (250 nM) supplemented with ATA (50 µM), MgCl 2 (12.5 mM), and 2 µM complementary DNA for 20 min in live-cell culture conditions. Unbound probes were removed by washing the cells using DPBS (3 times) and the reporter strands (Cy5 labeled H1 and H2, 250 nM each) diluted using opti-MEM (200 µL), supplemented with ATA (50 µM) and MgCl 2 (12.5 mM) were incubated with the cells for 60 min in live-cell culture conditions. Cells were finally washed with DPBS, exchanged the media with opti-MEM and proceed for live -cell confocal microscopy. The control studies were performed using cells lacking RNA target transfection, sensors, and only using H1 reporter. ψ -HCR was performed by transfection of Cy3-labeled 2 ′ -OMe modified extended RNA targets (T ′ -RNA, 60-nt) and choosing respective H1 and H2 reporters. A negative control for ψ -HCR was also performed by considering RNA target (T-RNA, 41-nt) lacking the extended split-initiator domain for HCR. Sample preparation for flow cytometry Live HEK293T and A549 cells were plated at 70-90% confluency into 6-well culture dish using DMEM media. The media was exchanged to opti-MEM (2 mL) prior to 1 h of RNA transfection. Cells were allowed to remain healthy prior transfection. Cy3-labeled 2 ′ -OMe modified RNA targets (100 nM) were mixed with 7.5 µL of P3000 reagent in a microcentrifuge tube and diluted with 75 µL opti-MEM media following mixed well. 5 µL Lipofectamine reagent was diluted with 75 µL opti-MEM media following mixed well. Both of these tubes were vortexed and centrifuged and combined into a single tube by vigorous mixing and allowed to stand for 15 min at room temperature. The cocktail mixture was added drop wise to the live cells and allowed for 60-90 min for transfection process. The cells were then washed thoroughly (3 times) using DPBS to remove the excess non-transfected targets. The cells were then trypsinized, centrifuged at 800 rpm for 4 min, and resuspended in opti-MEM (1 mL). The washing step was repeated twice and resuspended in opti-MEM (200 µL). Complementary DNA (4 µM, 40x excess) against RNA targets supplemented with ATA (50 µM) and MgCl 2 (12.5 mM) was incubated with the cells for 20 min at 4 °C for the complete quenching of extracellular RNA targets. The cells were then centrifuged, resuspended in opti-MEM (200 µL) and treated with HALOS (250 nM) supplemented with ATA (50 µM), MgCl 2 (12.5 mM), and 2 µM complementary DNA for 20 min in live-cell culture conditions. Unbound probes were removed by washing the cells using DPBS (3 times) and the reporter strands (Cy5 labeled H1 and H2, 250 nM each) diluted using opti-MEM (200 µL), supplemented with ATA (50 µM) and MgCl 2 (12.5 mM) were incubated with the cells for 60 min in live-cell culture conditions. The cells were then washed with DPBS (3 times) and resuspended in opti-MEM (500 µL) The cells were finally centrifuged, removed excess unreacted H1, H2 and exchanged the media with opti-MEM (500 µL) and proceed for flow-cytometry. The control studies were performed using cells lacking RNA targets, no HALOS, only using H1 reporter, following the same protocol mentioned above with HCR protocol. ( ψ -HCR) was performed by transfecting extended RNA targets (60-nt) and choosing respective H1 and H2. A negative control for ψ -HCR was also performed by considering RNA target (T-RNA, 41-nt) lacking the extended split-initiator domain for HCR.. Flow cytometry protocol The individually prepared samples were finally diluted to 500 µL opti-MEM media and subjected to flow cytometry studies. 10,000 events were collected from each sample and scanned using excitation of Cy3 (561 nm) and Cy5 (642 nm). The fluorescence scattering of the single cells was collected using respective band-pass filters. The live-cells were adequately gated from the single cell analysis and scattered plots of Cy3 vs. Cy5 intensity were generated. Nuclease and protease inhibition by ATA Aurintricarboxylic acid (ATA, 50 µM) was employed in live-cell HCR experiments to inhibit extracellular nuclease activity and maintain intracellular HALOS inserted domain and metastable hairpin monomers (H1, H2) stability. ATA is a broad-spectrum inhibitor of nucleases, including DNase I, exonucleases, and restriction endonucleases, acting by binding to positively charged regions of these enzymes and preventing access to DNA substrates, thereby blocking catalytic cleavage. 100 In vitro, ATA effectively inhibits DNase I activity at low micromolar concentrations (∼ 10 µM). During live-cell imaging, lower concentrations (∼ 10 µM) were insufficient to prevent degradation of single-stranded DNA reporters, necessitating an optimized concentration of 50 µM ATA. This concentration provides robust inhibition of both intra- and extracellular nucleases while minimizing cytotoxicity, which significantly increases above 100 µM and preserves cellular function over the assay duration. The reversible electrostatic and hydrogen bonding interactions of ATA with nucleases confer protection of nucleic acid probes, enabling reliable signal transduction without adverse effects on live cell viability. Supplementary Tables View this table: View inline View popup Download powerpoint Supplementary Table 1. Cholesterol labeled DNA hairpins. Cholesterol labeled DNA hairpin sequences used in the study. Internally modified Cholesterol is abbreviated as /iCholTEG/. View this table: View inline View popup Download powerpoint Supplementary Table 2. Cy3 labeled target DNA and RNA strands. Cy3 labeled DNA and OMe-modified RNA sequences used as target strands in the study. Cy3 modification at 3 ′ end is abbreviated as /3Cy3Sp/. View this table: View inline View popup Download powerpoint Supplementary Table 3. Complementary strands against target DNA and RNA. Complementary strand against Cy3-labeled DNA targets and Cy5-labeled reporter strands used in the study. View this table: View inline View popup Download powerpoint Supplementary Table 4. Cy5 labeled ssDNA reporters. Cy5-labeled ssDNA reporter sequences used in the study. Cy5 modification at 3 ′ and 5 ′ end are abbreviated as /3Cy5Sp/and /5Cy5/respectively. View this table: View inline View popup Download powerpoint Supplementary Table 5. Triangle reporter strands. DNA sequences used for the formation of triangle reporter.SCy5 modification at 3 ′ end is abbreviated as /3Cy5Sp/and biotin modification at 5 ′ end is abbreviated as /5BiotinTEG/. View this table: View inline View popup Download powerpoint Supplementary Table 6. Metastable hairpin monomers. DNA sequences used for the formation of triangle reporter.SCy5 modification at 3 ′ end is abbreviated as /3Cy5Sp/and biotin modification at 5 ′ end is abbreviated as /5BiotinTEG/. View this table: View inline View popup Download powerpoint Supplementary Table 7. Compositions and number of atoms to construct the membrane. Table of lipid compositions studied for membrane modeling and measuring their physical parameters using MD simulations. View this table: View inline View popup Download powerpoint Supplementary Table 8. HALOS variants and conformations studies using all-atom MD simulations. Table of HALOS variants and their conformations used for stability studies and signal transduction experiments using all-atom MD simulations. Supplementary Figures Download figure Open in new tab SFig. 1. Stability analysis of HALOS variants on membrane using all-atom MD simulations. (a) Schematics illustrate axial orientations of HALOS 14 and HALOS 31 within a POPC membrane. Heatmaps show the percentage of broken Watson–Crick base pairs in the paired stem domain during ∼1 µs simulations in aqueous solution and membrane environment.SBase-pair disruption was quantified over time using hydrogen bond criteria (A-T: <2 H-bonds; C-G: <3 H-bonds; distance cutoff 3.4 Å, angle cutoff 45 °C). HALOS 14 demonstrated greater base-pair disruption, indicating reduced structural stability compared to HALOS 31, suggesting HALOS 14 as a less suitable sensor variant. (b) Density distributions of the Rise base-pair step parameter reveal HALOS 31 maintains a sharp peak near 3.4 Å, consistent with canonical DNA spacing, while HALOS 14 deviates, reflecting structural distortion. Download figure Open in new tab SFig. 2. Additional confocal and flow cytometry analysis for HALOS variants in GUVs and live cells. In addition to Fig 1(g-h) , the membrane stability of HALOS variants with varying stem-length (21-28 bp) was evaluated in GUVS and live HEK293T cell.S (a) Schematic representation of HALOS variants differing in stem length and their membrane distribution in the axial orientation. (b-c) Confocal microscopy of HALOS stability in GUVs and live cell.SHALOS (250 nM) were incubated with GUVs and live cells in the absence of inside target (T-Cy3), followed by addition of reporter (R-Cy5, 100 nM) for 30 min. For imaging, GUVs were diluted five-fold in imaging buffer (1x TAE, 150 mM NaCl and 12.5 mM MgCl 2 ), and cells were washed with Opti-MEM medium containing 12.5 mM MgCl 2 and 50 µM ATA to remove unbound probe.SATTO488-encapsulated GUVs exhibited a gradual decrease in membrane Cy5 fluorescence with increasing stem length (21-28 bp), although HALOS 28 retained detectable signal intensity, indicating partial stem destabilization (b). In contrast, live cells showed significant Cy5 fluorescence for HALOS 21 but no detectable signal for HALOS 25-28, confirming improved membrane stability of longer-stem variants (c). Confocal images were acquired using 488 nm (ATTO488), 642 nm laser (Cy5) and halogen lamp (bright-field) under live-cell conditions (37 °C, 95% humidity, and 5% CO 2 ). (d-e) Flow cytometry of respective GUVs (d) and HEK293T cells (e) with and without (control) HALOS indicating similar trend of false-positive signal generation. (f) Percentage of HEK293T cells labeled with R-Cy5 (13-nt) upon HALOS treatment, depicting false positive signal.SScale bar: 10 µm (b) and 20 µm (c). Download figure Open in new tab SFig. 3. Additional MD simulations reveal membrane stability and signal transduction capability of HALOS and intermediate conformations. a Effect of the surrounding environment on the contour length of the paired region of the HALOS 31 stem, indicating less distortion in base pair stability for axially anchored HALOS compared to lateral and in aqueous solution. b Per-residue RMSF profiles of HALOS 31 demonstrate higher stability in the membrane compared to aqueous solution. c Representative intermediate structure shown from side and top views, along with Watson–Crick hydrogen bond patterns and cholesterol orientation distribution. d-f Comparative structural metrics for Axial, Intermediate, and Open hairpin conformations: (d) RMSD, (e) solvent-accessible surface area (SASA), and (f) radius of gyration (R g ). The Axial conformation, with the fewest unpaired bases, exhibits the lowest RMSD, SASA, and R g values, indicating the least deviations and compact structure in comparison to the other two. Download figure Open in new tab SFig. 4. NUPACK simulation and native PAGE analysis of HALOS-mediated signal transduction in solution. HALOS-mediated target recognition, hairpin opening, and reporter binding in solution were examined using NUPACK simulations 56 and native PAGE. (a) NUPACK-simulated structures of HALOS 31 resulting toehold-mediated T-Cy3 target binding, hairpin opening, and subsequent R-Cy5 recognition at 25 °C. Sequences were designed for thermodynamic stability. Target (T-Cy3) binding initiated at the toehold domain of HALOS and resulted in complete hairpin unfolding, enabling reporter (R-Cy5) hybridization. (b) HALOS (without cholesterol anchor) was annealed and incubated with an equimolar T-Cy3 for 15 min at room temperature, followed by R-Cy5 addition. A 10% native PAGE was run in 1x TBE buffer with 12.5 mM MgCl 2 at 100 V for 45 min at room temperature. Post-staining with EtBr (0.5 µg/mL, 10 min) and UV-imaging revealed T-Cy3 binding as a mobility shift versus HALOS alone, for both pre-annealed complexes (lane 3) and in situ detection (lane 4). R-Cy5 binding induced a further mobility shift with Cy5 fluorescence (lane 5). Without target or with scrambled target sequences, no stem opening or reporter labeling was observed (lanes 6–9). Download figure Open in new tab SFig. 5. Quenching of extravesicular targets and quantification of HALOS mediated target recognition. (a) Schematic illustration of quenching free extravesicular targets by incubation with complementary hairpin strands (∼20x molar excess) for 45–60 min at room temperature. (b-c) Quantification of HALOS 31–mediated target detection across GUV membrane.ST-Cy3 (250 nM) was encapsulated within GUVs (POPC) using electroformation, and excess external T-Cy3 was removed by centrifugation followed by treatment with complementary DNA (5 µM, ∼20× excess). The resulting GUVs were then incubated with HALOS 31 (250 nM) for 120 min at room temperature and analyzed by confocal microscopy (561 nm laser, Cy3). (b) The confocal images show Cy3 fluorescence localized to the membrane upon successful HALOS 31 target recognition, while failed recognition resulted in uniformly fluorescent interior.S(c) Detection efficiency was quantified as the percentage of ring-like fluorescence patterns (80–90%, n=90–100 GUVs across four fields of view). Scale bar: 20 µm (b). Download figure Open in new tab SFig. 6. Kinetics of HALOS 31-mediated target detection across GUV membranes. (a) Cy3-labeled target detection using HALOS 31 was monitored by tracking Cy3 ring formation over time across lipid bilayer.SComplementary DNA treated GUVs were plated onto a microscopy dishes, and time-lapse confocal imaging was initiated immediately after HALOS 31 addition. Time-lapse images were acquired for up to 120 min, and the percentage of GUVs exhibiting Cy3 ring patterns was calculated at each time point. Target detection appeared in a fraction of GUVs within 5 min, with additional events accumulating over time. (b) Quantification across over three fields of view showed a gradual increases in Cy3 ring formation from 19% at 5 min to 54% 120 min. Error bars were generated by bootstrapping Scale bar: 20 µm (a). Download figure Open in new tab SFig. 7. Additional large field of view confocal images and flow cytometry workflow for HALOS-mediated signal transduction. (a-b) Schematic and confocal images of controls lacking HALOS or containing HALOS without cholesterol anchors (b), showing no Cy3 membrane labeling of T-Cy3 encapsulated GUVs and absence of reporter signal on membrane. (c) Extended confocal view corresponding to Fig 2(b) , showing HALOS 31 mediated target recognition (Cy3) and signal transduction (Cy5). Most GUVs displayed membrane-associated Cy5 fluorescence consistent with signal transduction, while a minor population (<30%) exhibited R-Cy5 (13-nt ssDNA) diffusion across the membrane. (d) Schematic of flow cytometry analysis workflow used to generate histograms and Cy3 v.SCy5 scatter plot.S (e-g) Representative Cy3 v.SCy5 plots quantifying signal transduction efficiency: 27% for HALOS-mediated samples (e), 1.8% for samples without HALOS (b), and 0.01% for GUVs lacking encapsulated targets (g). Scale bar: 20 µm (a-c), 5 µm (cropped images from c). Download figure Open in new tab SFig. 8. HALOS-mediated outside-in signaling mimicking GPCR pathways and evaluation of live-cell compatibility. (a-b) To mimic GPCR signaling on synthetic vesicles, outside-in signal transduction were performed using HALOS 31. GUVs encapsulated with R-Cy5 were treated with HALOS 31 following quenching of external R-Cy5 by complementary DNA. Further, addition of T-Cy3 (GPCR-ligand mimic) to the external medium allowed recruitment to the outward-facing toehold of HALOS 31, triggering hairpin opening and subsequent signal transduction by internal recognition of intravesicular R-Cy5. (b) Formation of Cy3 and Cy5 rings at the GUV membrane confirmed T-Cy3 recognition by HALOS, indicating successful signal transduction. (c) In the absence of external ligand, no Cy5 ring formation was observed, demonstrating no signal transmission. (d) To assess live-cell compatibility, HEK293T cells were incubated with HALOS 31 (250 nM) in Opti-MEM containing 50 µM ATA and 12.5 mM MgCl 2 for 15 min under live-cell culture environment. After washing, cells were sequentially treated to T-Cy3 (250 nM) and R-Cy5 (250 nM) with washing steps between each addition. Confocal imaging showed both Cy3 and Cy5 fluorescence at the cell membrane, demonstrating compatibility of HALOS-mediated signal transduction on live-cell membrane. Scale bar: 20 µm (b-d). Download figure Open in new tab SFig. 9. Additional large-field-of-view images of scramble controls. (a) Schematic and confocal images of GUVs containing scramble targets (Scr. T-Cy3, 250 nM) treated with HALOS 31 show no Cy3 ring formation on membrane. Addition of 13-nt ssDNA reporter (R-Cy5, 100 nM) resulted no Cy5 ring formation, confirming no signal transduction across membrane. (b) Cy3 v.SCy5 flow cytometry plots reveal minimal signal transduction efficiency (0.61%) across membrane for scramble control.S (c–d) GUVs encapsulating sequence-specific targets (T-Cy3, 250 nM) form Cy3 rings upon HALOS 31 treatment, but display no membrane Cy5 labeling in confocal microscopy and minimal efficiency (0.30%) in flow cytometry with polyT scramble reporter (100 nM), indicating sequence-specificity in HALOS-mediated signal transduction. Scale bar: 20 µm (a,c). Download figure Open in new tab SFig. 10. Bulkier reporter design for leakless signal transduction. (a) Schematic of gradient-driven leakage of the 13-nt ssDNA reporter (R-Cy5) across membranes during signal transduction, where HALOS-driven toroidal nanopore formation 38 , causes false positive signals by reporter diffusion and nonspecific labeling to overhanged T-Cy3. (b) Design of a bulkier triangular reporter (Tr) formed by two complementary DNA strands extending reporter sequence to 21-nt, labeled with Cy5 and biotin for streptavidin (SA) anchoring to increase steric bulkines.S (c) NUPACK simulation confirms thermodynamic stability of Tr binding to open HALOS conformation. (d) Native 10% PAGE analysis shows formation of Tr reporter complexes with slower mobility than monomers and further shifted bands upon binding to open conformation of HALO.SSpecificity in binding was confirmed by control interactions with HALOS alone and scramble reporter sequence.S (e-h) Additional large-field confocal images (presented in Fig 3(d) ) of SA-TrCy5 signaling from T-Cy3 encapsulated GUVs show no reporter leakage inside GUV.SControl GUVs (ATTO488) show no signal transduction, confirming labeling specificity. Scale bar: 20 µm (e-h). Download figure Open in new tab SFig. 11. All-atom MD simulations reveal physical properties of lipid membranes. (a) Chemical structures of POPC (and, EggPC) and LPC, alongside, MD simulation snapshot of mixed POPC+LPC membrane. (b) Head group density profiles along the Z-axis of all lipid compositions studied, indicating membrane thicknes.S (c) Deuterium order parameters for the two acyl chains across all five models, reflecting membrane fluidity. (c) Area per lipid measurements for each membrane model, indicating membrane packing Download figure Open in new tab SFig. 12. Membrane fluidity affects HALOS-mediated signal transduction in GUVs. (a-b) Membrane polarity was assessed by calculating Laurdan generalized polarization (GP) value.SGUVs of various lipid composition were incubated with Laurdan (5 µM for 15 min) and fluorescence spectra (a) were recorded to calculate GP values (b) by considering the fluorescence intensity at 436 nm (I B ) and 512 nm (I R ) upon excitation at 390 nm. Three replicates were measured for each lipid compositions in this study. (c) GP images were generated from confocal images at these wavelengths using a custom ImageJ plugin, showing lipid-composition dependent GP shift.S (d) Spectral scanning of Laurdan indicated fluidity changes between (POPC+30% Chol) and (30% LPC + POPC) membranes; excitation: 405 nm, emission: 420-600 nm with 10 nm step size. (e-f) Confocal imaging of target recognition (Cy3) and signal transduction (Cy5) in GUVs of different lipid composition demonstrated increased signal transduction with elevated membrane fluidity by higher LPC component. (g-k) Laurdan emission spectra of GUVs with and without HALOS (250 nM) confirmed membrane properties remain largely unchanged upon HALOS addition. Scale bar: 5 µm (c-d), 20 µm (e-f) Download figure Open in new tab SFig. 13. Additional large Field-of-view images of HALOS-mediated long extended-DNA and RNA-target recognition in GUVs. (a-c) Extended confocal-view corresponding to Fig 2(b) , GUVs were encapsulated with longer DNA targets (100-nt, 250 nM) while keeping the recognition domain (41-nt) with polyT overhang SUpon treatment with HALOS 31 (250 nM), all three isomers of long extended-targets (extended 1-3) showed Cy3 ring formation indicating target recognition independent of position of target recognition domain. Subsequent addition of Cy5-TrStp reporters (100 nM), yielded Cy5 ring formation, suggesting signal transduction ability of these long-DNA target.S d Encapsulation and detection of 2 ′ -OMe-RNA (41 nt, 250 nm) in GUVs by HALOS, confirmed by Cy3 and Cy5 membrane rings, verifying efficient RNA target detection across membrane. Scale bar: 20 µm (a-d). Download figure Open in new tab SFig. 14. NUPACK simulation and Agarose gel analysis of HALOS-mediated hybridization chain reaction in solution. (a) Target triggered hairpin opening in HALOS and initiation of hybridization chain reaction on open hairpin (initiator). NUPACK simulated structure of metastable hairpin monomers (H1, H2) coupled structure on HALOS depicting spontaneous opening of hairpin monomers in the presence of initiator and thermodynamic stability at 25 °C. Sequences of H1 and H2 were designed with maximum thermodynamic energy gain with no cross-reactivity. (b) Agarose gel electrophoresis of HCR in solution (1x TAE+150 mM NaCl+12.5 mM MgCl 2 ) supports the initiation of HCR with initiator (HALOS 31+T-Cy3). The HCR progress was monitored with varied ratio of initiator (1 to 0.1 equiv.), while no HCR was observed in the absence of T-Cy3 and HALOS 31. 1% (w/v) Agarose gel electrophoresis was performed using 1× Sodium Borate buffer (10 mM NaOH, pH 8.5) supplemented with 12.5 mM MgCl 2 . The gel was prestained with SYBR gold (0.5 µg/mL), loaded with 10 pmol (1 µM x 10 µL) sample and run with 100 V for 90 min under ice-cold condition and imaged by UV transillumination. Download figure Open in new tab SFig. 15. Hybridization chain reaction amplifies target-specific signal in GUVs. (a-b) Extended confocal views demonstrate HCR-based signal amplification from target-encapsulated (T-Cy3) GUVs, while control GUVs (ATTO488) show no amplification (a). Reporter H1 yields low signal-to-noise compared to HCR as illustrated by confocal images and Cy5 intensity profiles (b). (c) Cy3 v.SCy5 scatter plots compare HCR reactions with H1 only and controls lacking HALOS 31 and target (T-Cy3), revealing higher Cy5 signal for HCR, compared to H1 alone and negative control.S (d-e) 3D view of GUVs after HCR on the membrane. GUVs containing Cy3 target, treated with HALOS 31 and HCR monomers, were subjected to 3D confocal microscopy over 15 µm depth with 500 nm z-stack resolution. The 3D images were reconstructed using ImageJ 3D viewer plugin. The homogeneous distribution of Cy3 and Cy5 labeling on GUVs was confirmed by different angle of view (YZ and XZ plane), confirming no non-specific labeling of HCR monomers to GUV.SScale bar: 20 µm (a-b), 5 µm (d-e). Download figure Open in new tab SFig. 16. Signal amplification of HALOS-mediated RNA detection in live A549 cells. (a-f) Additional confocal microscopy images for signal amplification for HALOS-mediated RNA detection and amplification in live A549 cell.SCells were transfected with 2 ′ -OMe-RNA target (41-nt) using lipofectamine-based transfection. Negative controls lacking HALOS, target RNA, or both displayed no membrane Cy3 and Cy5 fluorescence (a–c). The target RNA transfected cells treated with HALOS 31 resulted bright Cy5 signal upon performing HCR reaction (60 min in cell-culture environment at 37 °C) with substantially higher Cy5 intensity for HCR versus H1 alone (d-e). HALOS having mismatched sequence (HALOS 14) with target RNA resulted no signal transduction and amplification (f). Flow cytometry Cy3 v.SCy5 scatter plots corroborate amplified Cy5 surface signal for HCR compared to H1 and negative control.SScale bar: 20 µm (a–f). Download figure Open in new tab SFig. 17. NUPACK simulation and native PAGE confirms dual reporter labeling of extended target treated HALOS 31 in solution. (a-c) NUPACK simulated structure of extended target (T ′ -Cy3, 60-nt) bound to HALOS 31 (a), linear and SA-Tr dual-reporter (Cy5 and AF488) labeling to the opened hairpins (b-c), confirming thermodynamic stability and no cross reactivity between reporter.S (b) Native PAGE of dual reporter labeling to open HALOS conformation in solution. 10% Native PAGE was run using 1x TBE buffer + 12.5 mM MgCl 2 at 100 V for 45 min at room temperature. The gel was post-stained using EtBr (0.5 µg/mL) for 10 min and imaged using UV-transillumination. The fluorescence images were scanned using Image Quant 800 gel imager. Addition of both R-Cy5 and R-AF488 resulted in dual labeling of T ′ -Cy3 (60 nt) treated HALOS 31 (lane 2). Absence of either extension from extend domain from target (T-Cy3, 41 nt) or HALOS (lacking open hairpin domain) showed no labeling of R-AF488 and R-Cy5 respectively(lane 3-4), confirming no cross reactivity between R-Cy5 and AF488 reporter.SThe single color reporter (either R-Cy5 or R-AF488) labeling to T ′ -Cy3 (60 nt) treated HALOS 31 resulted in specific reporter labeling as depicted by fluorescence image (lane 7-8). In the absence of target (T ′ -Cy3), HALOS 31 remained uncreative to any reporters (lane 9). Download figure Open in new tab SFig. 18. Additional dual reporter labeling images in GUVs and live-cell compatibility. (a) Extended-view confocal images for dual reporter labeling in GUV.SWhen treated with R-Cy5 (100 nM) or R-AF488 (100 nM) individually, T ′ -Cy3 detected GUVs showed specific fluorescence on the respective Cy3 or AF488 channel; both were observed when both reporters were applied simultaneously. (b) Dual reporter labeling with control GUVs (Coumarin, Cyan). Cy3 target GUVs display dual reporter labeling while no reporter labeling on coumarin GUVs, suggesting no-non-specific labeling (c) Live-cell compatibility of dual reporter probe.SLive HEK293T cells were incubated with HALOS 31 (250 nM) in Opti-MEM containing 50 µM ATA and 12.5 mM MgCl 2 for 15 min under live-cell culture environment. After washing, cells were sequentially treated to T-Cy3 (250 nM) and R-Cy5 + R-AF488 (250 nM each) with washing steps (3x) between each addition. Confocal imaging showed Cy3, Cy5 and AF488 fluorescence at the cell membrane, demonstrating compatibility of HALOS-mediated signal transduction using dual reporter labeling on live-cell membrane. Scale bar: 20 µm (a-c). Download figure Open in new tab SFig. 19. NUPACK simulation and native PAGE analysis of ψ -HCR-mediated signal amplification. (a) Extended target (T ′ -Cy3, 60-nt) triggered hairpin opening in HALOS and initiation of ψ -HCR by proximity induced split-initiator generation. NUPACK simulated structure of metastable hairpin monomers (H1, H2) coupled structure on HALOS depicting spontaneous opening of hairpin monomers in the presence of split-initiator and their thermodynamic stability at 25 °C. Sequences of H1 and H2 were designed with maximum thermodynamic energy gain with no cross-reactivity. (b) Native PAGE of ψ -HCR in solution (1x TAE+150 mM NaCl+12.5 mM MgCl 2 ) supports the initiation of HCR with initiator (HALOS 31+T ′ -Cy3). The HCR progress was monitored with varied ratio of initiator (1 to 0.25 equiv.), while no HCR was observed in the absence of T ′ -Cy3 and HALOS 31. negative controls lacking either split initiator (SI) 1 (extended target domain) or 2 (open hairpin domain) resulted no ψ -HCR product formation, confirming requirement of both the split initiators for reaction. A 10% native PAGE was run in 1x TBE buffer with 12.5 mM MgCl 2 at 100 V for 60 min at room temperature, Post-stained with EtBr (0.5 µg/mL, 10 min) and imaged using UV-transillumination. Download figure Open in new tab SFig. 20. Signal amplification in GUVs confirms proximity-based initiation of ψ -HCR in GUVs. (a-c) Confocal images demonstrate ψ -HCR-based signal amplification from extended target-encapsulated (T ′ -Cy3) GUV.SControl GUVs (ATTO488) and GUVs with non-extended target (T-Cy3, 41 nt) confirming the non-leaky behavior of ψ -HCR monomers on non-target GUVs (a). With extended target (T ′ -Cy3, 60 nt), reporter H1 produced low signal-to-noise, while ψ -HCR enabled pronounced signal amplification, confirming proximity induced initiation of HCR (b-c). Scale bar: 20 µm (a-c). Download figure Open in new tab SFig. 21. Signal amplification of HALOS-mediated RNA detection in live A549 cells using ψ -HCR. (a-f) Additional confocal microscopy images for signal amplification for HALOS-mediated RNA detection and amplification using ψ -HCR in live A549 cell.SCells were transfected with 2 ′ -OMe-RNA target (2 ′ -OMe-T ′ -Cy3, 60-nt) using lipofectamine-based transfection. Negative controls lacking HALOS, target RNA, or both displayed no membrane Cy3 and Cy5 fluorescence (a–c). Further, in the absence of extended domain in target RNA (2 ′ -OMe-T-Cy3„ 41-nt), showing target detection using membrane Cy3 labeling but without amplified Cy5 signal, indicating failure to initiate ψ -HCR (d). The extended target RNA (60-nt) transfected cells treated with HALOS 31 resulted bright Cy5 signal upon performing ψ -HCR reaction (60 min in cell-culture environment at 37 °C) with substantially higher Cy5 intensity for HCR versus H1 alone (e-f). Flow cytometry Cy3 v.SCy5 scatter plots corroborate amplified Cy5 surface signal for HCR compared to H1 and negative control.SScale bar: 20 µm (a–f). Acknowledgments The authors thank E. Winfree, L. Qian, A. Aksimentiev, D. Karna, P. Chopade, and Y. Hassan for their insightful discussions and valuable comments on our work. H.J. thanks supercomputer facility for providing access through the NSM PARAM SEVA at IIT Hyderabad, India. Funder Information Declared NIH , 1DP2AI144247 , 1R61CA278558 Flinn Foundation, https://ror.org/058qtek75 , 24-17692 SERB , SRG/2022/002109 DST Inspire Faculty Fellowship, India , IFA20- PH-256 American Heart Association, https://ror.org/013kjyp64 , 23PRE1029870 References 1. ↵ Levental I , Lyman E ( 2023 ) Regulation of membrane protein structure and function by their lipid nano-environment . Nat. Rev. Mol. Cell Biol . 24 ( 2 ): 107 – 122 . OpenUrl CrossRef PubMed 2. Su J , et al. ( 2024 ) Cell–cell communication: new insights and clinical implications . Signal Transduct. Target. Ther . 9 ( 1 ): 196 . OpenUrl PubMed 3. ↵ Groves JT , Kuriyan J ( 2010 ) Molecular mechanisms in signal transduction at the membrane . Nat. Struct. Mol. Biol 17 ( 6 ): 659 – 665 . OpenUrl CrossRef PubMed Web of Science 4. ↵ Zhang M , et al. ( 2024 ) G protein-coupled receptors (GPCRs): advances in structures, mechanisms and drug discovery . Signal Transduction Targeted Ther . 9 ( 1 ): 88 . 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