Mechanism of single-stranded DNA annealing by RAD52-RPA complex.

RAD52 is important for the repair of DNA double-stranded breaks1,2, mitotic DNA synthesis3-5 and alternative telomere length maintenance6,7. Central to these functions, RAD52 promotes the annealing of complementary single-stranded DNA (ssDNA)8,9 and provides an alternative to BRCA2/RAD51-dependent homologous recombination repair10. Inactivation of RAD52 in homologous-recombination-deficient BRCA1- or BRCA2-defective cells is synthetically lethal11,12, and aberrant expression of RAD52 is associated with poor cancer prognosis13,14. As a consequence, RAD52 is an attractive therapeutic target against homologous-recombination-deficient breast, ovarian and prostate cancers15-17. Here we describe the structure of RAD52 and define the mechanism of annealing. As reported previously18-20, RAD52 forms undecameric (11-subunit) ring structures, but these rings do not represent the active form of the enzyme. Instead, cryo-electron microscopy and biochemical analyses revealed that ssDNA annealing is driven by RAD52 open rings in association with replication protein-A (RPA). Atomic models of the RAD52-ssDNA complex show that ssDNA sits in a positively charged channel around the ring. Annealing is driven by the RAD52 N-terminal domains, whereas the C-terminal regions modulate the open-ring conformation and RPA interaction. RPA associates with RAD52 at the site of ring opening with critical interactions occurring between the RPA-interacting domain of RAD52 and the winged helix domain of RPA2. Our studies provide structural snapshots throughout the annealing process and define the molecular mechanism of ssDNA annealing by the RAD52-RPA complex.

Rapid Long-distance Migration of RPA on Single Stranded DNA Occurs Through Intersegmental Transfer Utilizing Multivalent Interactions.

Replication Protein A (RPA) is asingle strandedDNA(ssDNA)binding protein that coordinates diverse DNA metabolic processes including DNA replication, repair, and recombination. RPA is a heterotrimeric protein with six functional oligosaccharide/oligonucleotide (OB) domains and flexible linkers. Flexibility enables RPA to adopt multiple configurations andis thought to modulate its function. Here, usingsingle moleculeconfocal fluorescencemicroscopy combinedwith optical tweezers and coarse-grained molecular dynamics simulations, we investigated the diffusional migration of single RPA molecules on ssDNA undertension.The diffusioncoefficientDis the highest (20,000nucleotides2/s) at 3pNtension and in 100 mMKCl and markedly decreases whentensionor salt concentrationincreases. We attribute the tension effect to intersegmental transfer which is hindered by DNA stretching and the salt effect to an increase in binding site size and interaction energy of RPA-ssDNA. Our integrative study allowed us to estimate the size and frequency of intersegmental transfer events that occur through transient bridging of distant sites on DNA by multiple binding sites on RPA. Interestingly, deletion of RPA trimeric core still allowed significant ssDNA binding although the reduced contact area made RPA 15-fold more mobile. Finally, we characterized the effect of RPA crowding on RPA migration. These findings reveal how the high affinity RPA-ssDNA interactions are remodeled to yield access, a key step in several DNA metabolic processes.

Phase separation properties of RPA combine high-affinity ssDNA binding with dynamic condensate functions at telomeres.

RPA has been shown to protect single-stranded DNA (ssDNA) intermediates from instability and breakage. RPA binds ssDNA with sub-nanomolar affinity, yet dynamic turnover is required for downstream ssDNA transactions. How ultrahigh-affinity binding and dynamic turnover are achieved simultaneously is not well understood. Here we reveal that RPA has a strong propensity to assemble into dynamic condensates. In solution, purified RPA phase separates into liquid droplets with fusion and surface wetting behavior. Phase separation is stimulated by sub-stoichiometric amounts of ssDNA, but not RNA or double-stranded DNA, and ssDNA gets selectively enriched in RPA condensates. We find the RPA2 subunit required for condensation and multi-site phosphorylation of the RPA2 N-terminal intrinsically disordered region to regulate RPA self-interaction. Functionally, quantitative proximity proteomics links RPA condensation to telomere clustering and integrity in cancer cells. Collectively, our results suggest that RPA-coated ssDNA is contained in dynamic RPA condensates whose properties are important for genome organization and stability.

Rtt105 regulates RPA function by configurationally stapling the flexible domains.

Replication Protein A (RPA) is a heterotrimeric complex that binds to single-stranded DNA (ssDNA) and recruits over three dozen RPA-interacting proteins to coordinate multiple aspects of DNA metabolism including DNA replication, repair, and recombination. Rtt105 is a molecular chaperone that regulates nuclear localization of RPA. Here, we show that Rtt105 binds to multiple DNA binding and protein-interaction domains of RPA and configurationally staples the complex. In the absence of ssDNA, Rtt105 inhibits RPA binding to Rad52, thus preventing spurious binding to RPA-interacting proteins. When ssDNA is available, Rtt105 promotes formation of high-density RPA nucleoprotein filaments and dissociates during this process. Free Rtt105 further stabilizes the RPA-ssDNA filaments by inhibiting the facilitated exchange activity of RPA. Collectively, our data suggest that Rtt105 sequesters free RPA in the nucleus to prevent untimely binding to RPA-interacting proteins, while stabilizing RPA-ssDNA filaments at DNA lesion sites.

Generation of versatile ss-dsDNA hybrid substrates for single-molecule analysis.

Here, we describe a rapid and versatile protocol to generate gapped DNA substrates for single-molecule (SM) analysis using optical tweezers via site-specific Cas9 nicking and force-induced melting. We provide examples of single-stranded (ss) DNA gaps of different length and position. We outline protocols to visualize these substrates by replication protein A-enhanced Green Fluorescent Protein (RPA-eGFP) and SYTOX Orange staining using commercially available optical tweezers (C-TRAP). Finally, we demonstrate the utility of these substrates for SM analysis of bidirectional growth of RAD-51-ssDNA filaments. For complete details on the use and execution of this protocol, please refer to Belan et al. (2021).

SETD2-mediated H3K14 trimethylation promotes ATR activation and stalled replication fork restart in response to DNA replication stress.

Ataxia telangiectasia and Rad3 related (ATR) activation after replication stress involves a cascade of reactions, including replication protein A (RPA) complex loading onto single-stranded DNA and ATR activator loading onto chromatin. The contribution of histone modifications to ATR activation, however, is unclear. Here, we report that H3K14 trimethylation responds to replication stress by enhancing ATR activation. First, we confirmed that H3K14 monomethylation, dimethylation, and trimethylation all exist in mammalian cells, and that both SUV39H1 and SETD2 methyltransferases can catalyze H3K14 trimethylation in vivo and in vitro. Interestingly, SETD2-mediated H3K14 trimethylation markedly increases in response to replication stress induced with hydroxyurea, a replication stress inducer. Under these conditions, SETD2-mediated H3K14me3 recruited the RPA complex to chromatin via a direct interaction with RPA70. The increase in H3K14me3 levels was abolished, and RPA loading was attenuated when SETD2 was depleted or H3K14 was mutated. Rather, the cells were sensitive to replication stress such that the replication forks failed to restart, and cell-cycle progression was delayed. These findings help us understand how H3K14 trimethylation links replication stress with ATR activation.

Generation of Fluorescent Versions of Saccharomyces cerevisiae RPA to Study the Conformational Dynamics of Its ssDNA-Binding Domains.

Replication protein A (RPA) is an essential single-stranded DNA (ssDNA)-binding protein that sequesters ssDNA and protects it from nucleolytic degradation. The RPA-ssDNA nucleoprotein acts as a hub to recruit over two dozen DNA metabolic enzymes onto ssDNA to coordinate DNA replication, repair, and recombination. RPA functions as a heterotrimer composed of RPA70, RPA32, and RPA14 subunits and has multiple DNA-binding and protein-interaction domains. Several of these domains are connected by disordered linkers allowing RPA to adopt a wide variety of conformations on ssDNA. Here we describe a fluorescence-based tool to monitor the dynamics of select DNA-binding domains of RPA. Noncanonical amino acids are utilized to site-specifically engineer fluorescent probes in Saccharomyces cerevisiae RPA heterologously expressed in BL21 (DE3) and its derivatives. A procedure to synthesize 4-azido-L-phenylalanine (4AZP), a noncanonical amino acid, is also described. Sites for fluorophore positioning that produce a measurable change in fluorescence upon binding to ssDNA are detailed. This fluorescence enhancement through noncanonical amino acid (FEncAA) approach can also be applied to other DNA-binding proteins to investigate the dynamics of protein-nucleic acid interactions.

RPA-1 from Leishmania sp.: Recombinant Protein Expression and Purification, Molecular Modeling, and Molecular Dynamics Simulations Protocols.

RPA is a conserved heterotrimeric complex and the major single-stranded DNA (ssDNA)-binding protein heterotrimeric complex, which in eukaryotes is formed by the RPA-1, RPA-2, and RPA-3 subunits. The main structural feature of RPA is the presence of the oligonucleotide/oligosaccharide-binding fold (OB-fold) domains, responsible for ssDNA binding and protein:protein interactions. Among the RPA subunits, RPA-1 bears three of the four OB folds involved with RPA-ssDNA binding, although in some organisms RPA-2 can also bind ssDNA. The OB-fold domains are also present in telomere end-binding proteins (TEBP), essential for chromosome end protection. RPA-1 from Leishmania sp., as well as RPA-1 from trypanosomatids, a group of early-divergent protozoa, shows some structural differences compared to higher eukaryote RPA-1. Also, RPA-1 from Leishmania sp., similar to TEBPs, may exert telomeric protective functions. Remarkably, different pieces of evidence have pointed out that trypanosomatids may not have OB fold-containing TEBPs. Moreover, recent data indicate that trypanosomatid RPA-1 may be considered a TEBP since it shares with TEBPs conserved functional and structural features. However, it is still unknown whether the RPA-1 protective telomeric role is exclusive to trypanosomatids or is also present in other primitive eukaryotes. Here, we describe a protocol to obtain highly purified and biologically active Leishmania amazonensis recombinant RPA-1, and to perform molecular modeling and molecular dynamics simulations methods which could be probably applied to functional and structural studies of homologous proteins in other primitive eukaryotes.

KERA: analysis tool for multi-process, multi-state single-molecule data.

Molecular machines within cells dynamically assemble, disassemble and reorganize. Molecular interactions between their components can be observed at the single-molecule level and quantified using colocalization single-molecule spectroscopy, in which individual labeled molecules are seen transiently associating with a surface-tethered partner, or other total internal reflection fluorescence microscopy approaches in which the interactions elicit changes in fluorescence in the labeled surface-tethered partner. When multiple interacting partners can form ternary, quaternary and higher order complexes, the types of spatial and temporal organization of these complexes can be deduced from the order of appearance and reorganization of the components. Time evolution of complex architectures can be followed by changes in the fluorescence behavior in multiple channels. Here, we describe the kinetic event resolving algorithm (KERA), a software tool for organizing and sorting the discretized fluorescent trajectories from a range of single-molecule experiments. KERA organizes the data in groups by transition patterns, and displays exhaustive dwell time data for each interaction sequence. Enumerating and quantifying sequences of molecular interactions provides important information regarding the underlying mechanism of the assembly, dynamics and architecture of the macromolecular complexes. We demonstrate KERA's utility by analyzing conformational dynamics of two DNA binding proteins: replication protein A and xeroderma pigmentosum complementation group D helicase.

Single molecule microscopy reveals key physical features of repair foci in living cells.

In response to double strand breaks (DSB), repair proteins accumulate at damaged sites, forming membrane-less sub-compartments or foci. Here we explored the physical nature of these foci, using single molecule microscopy in living cells. Rad52, the functional homolog of BRCA2 in yeast, accumulates at DSB sites and diffuses ~6 times faster within repair foci than the focus itself, exhibiting confined motion. The Rad52 confinement radius coincides with the focus size: foci resulting from 2 DSBs are twice larger in volume that the ones induced by a unique DSB and the Rad52 confinement radius scales accordingly. In contrast, molecules of the single strand binding protein Rfa1 follow anomalous diffusion similar to the focus itself or damaged chromatin. We conclude that while most Rfa1 molecules are bound to the ssDNA, Rad52 molecules are free to explore the entire focus reflecting the existence of a liquid droplet around damaged DNA.

Hydrogen-deuterium exchange reveals a dynamic DNA-binding map of replication protein A.

Replication protein A (RPA) binds to single-stranded DNA (ssDNA) and interacts with over three dozen enzymes and serves as a recruitment hub to coordinate most DNA metabolic processes. RPA binds ssDNA utilizing multiple oligosaccharide/oligonucleotide binding domains and based on their individual DNA binding affinities are classified as high versus low-affinity DNA-binding domains (DBDs). However, recent evidence suggests that the DNA-binding dynamics of DBDs better define their roles. Utilizing hydrogen-deuterium exchange mass spectrometry (HDX-MS), we assessed the ssDNA-driven dynamics of the individual domains of human RPA. As expected, ssDNA binding shows HDX changes in DBDs A, B, C, D and E. However, DBD-A and DBD-B are dynamic and do not show robust DNA-dependent protection. DBD-C displays the most extensive changes in HDX, suggesting a major role in stabilizing RPA on ssDNA. Slower allosteric changes transpire in the protein-protein interaction domains and linker regions, and thus do not directly interact with ssDNA. Within a dynamics-based model for RPA, we propose that DBD-A and -B act as the dynamic half and DBD-C, -D and -E function as the less-dynamic half. Thus, segments of ssDNA buried under the dynamic half are likely more readily accessible to RPA-interacting proteins.

PCNA Monoubiquitination Is Regulated by Diffusion of Rad6/Rad18 Complexes along RPA Filaments.

Translesion DNA synthesis (TLS) enables DNA replication through damaging modifications to template DNA and requires monoubiquitination of the proliferating cell nuclear antigen (PCNA) sliding clamp by the Rad6/Rad18 complex. This posttranslational modification is critical to cell survival following exposure to DNA-damaging agents and is tightly regulated to restrict TLS to damaged DNA. Replication protein A (RPA), the major single-strand DNA (ssDNA) binding protein complex, forms filaments on ssDNA exposed at TLS sites and plays critical yet undefined roles in regulating PCNA monoubiquitination. Here, we utilize kinetic assays and single-molecule FRET microscopy to monitor PCNA monoubiquitination and Rad6/Rad18 complex dynamics on RPA filaments, respectively. Results reveal that a Rad6/Rad18 complex is recruited to an RPA filament via Rad18·RPA interactions and randomly translocates along the filament. These translocations promote productive interactions between the Rad6/Rad18 complex and the resident PCNA, significantly enhancing monoubiquitination. These results illuminate critical roles of RPA in the specificity and efficiency of PCNA monoubiquitination and represent, to the best of our knowledge, the first example of ATP-independent translocation of a protein complex along a protein filament.

Determinants of replication protein A subunit interactions revealed using a phosphomimetic peptide.

Replication protein A (RPA) is a eukaryotic ssDNA-binding protein and contains three subunits: RPA70, RPA32, and RPA14. Phosphorylation of the N-terminal region of the RPA32 subunit plays an essential role in DNA metabolism in processes such as replication and damage response. Phosphorylated RPA32 (pRPA32) binds to RPA70 and possibly regulates the transient RPA70-Bloom syndrome helicase (BLM) interaction to inhibit DNA resection. However, the structural details and determinants of the phosphorylated RPA32-RPA70 interaction are still unknown. In this study, we provide molecular details of the interaction between RPA70 and a mimic of phosphorylated RPA32 (pmRPA32) using fluorescence polarization and NMR analysis. We show that the N-terminal domain of RPA70 (RPA70N) specifically participates in pmRPA32 binding, whereas the unphosphorylated RPA32 does not bind to RPA70N. Our NMR data revealed that RPA70N binds pmRPA32 using a basic cleft region. We also show that at least 6 negatively charged residues of pmRPA32 are required for RPA70N binding. By introducing alanine mutations into hydrophobic positions of pmRPA32, we found potential points of contact between RPA70N and the N-terminal half of pmRPA32. We used this information to guide docking simulations that suggest the orientation of pmRPA32 in complex with RPA70N. Our study demonstrates detailed features of the domain-domain interaction between RPA70 and RPA32 upon phosphorylation. This result provides insight into how phosphorylation tunes transient bindings between RPA and its partners in DNA resection.

Replication protein A binds RNA and promotes R-loop formation.

Replication protein A (RPA), a major eukaryotic ssDNA-binding protein, is essential for all metabolic processes that involve ssDNA, including DNA replication, repair, and damage signaling. To perform its functions, RPA binds ssDNA tightly. In contrast, it was presumed that RPA binds RNA weakly. However, recent data suggest that RPA may play a role in RNA metabolism. RPA stimulates RNA-templated DNA repair in vitro and associates in vivo with R-loops, the three-stranded structures consisting of an RNA-DNA hybrid and the displaced ssDNA strand. R-loops are common in the genomes of pro- and eukaryotes, including humans, and may play an important role in transcription-coupled homologous recombination and DNA replication restart. However, the mechanism of R-loop formation remains unknown. Here, we investigated the RNA-binding properties of human RPA and its possible role in R-loop formation. Using gel-retardation and RNA/DNA competition assays, we found that RPA binds RNA with an unexpectedly high affinity (KD ≈ 100 pm). Furthermore, RPA, by forming a complex with RNA, can promote R-loop formation with homologous dsDNA. In reconstitution experiments, we showed that human DNA polymerases can utilize RPA-generated R-loops for initiation of DNA synthesis, mimicking the process of replication restart in vivo These results demonstrate that RPA binds RNA with high affinity, supporting the role of this protein in RNA metabolism and suggesting a mechanism of genome maintenance that depends on RPA-mediated DNA replication restart.

The structure of human CST reveals a decameric assembly bound to telomeric DNA.

The CTC1-STN1-TEN1 (CST) complex is essential for telomere maintenance and resolution of stalled replication forks genome-wide. Here, we report the 3.0-angstrom cryo-electron microscopy structure of human CST bound to telomeric single-stranded DNA (ssDNA), which assembles as a decameric supercomplex. The atomic model of the 134-kilodalton CTC1 subunit, built almost entirely de novo, reveals the overall architecture of CST and the DNA-binding anchor site. The carboxyl-terminal domain of STN1 interacts with CTC1 at two separate docking sites, allowing allosteric mediation of CST decamer assembly. Furthermore, ssDNA appears to staple two monomers to nucleate decamer assembly. CTC1 has stronger structural similarity to Replication Protein A than the expected similarity to yeast Cdc13. The decameric structure suggests that CST can organize ssDNA analogously to the nucleosome's organization of double-stranded DNA.

A multi-approach analysis highlights the relevance of RPA-1 as a telomere end-binding protein (TEBP) in Leishmania amazonensis.

BACKGROUND: Telomeres are chromosome end structures important in the maintenance of genome homeostasis. They are replenished by the action of telomerase and associated proteins, such as the OB (oligonucleotide/oligosaccharide-binding)-fold containing telomere-end binding proteins (TEBP) which plays an essential role in telomere maintenance and protection. The nature of TEBPs is well known in higher and some primitive eukaryotes, but it remains undetermined in trypanosomatids. Previous in silico searches have shown that there are no homologs of the classical TEPBs in trypanosomatids, including Leishmania sp. However, Replication Protein A subunit 1 (RPA-1), an OB-fold containing DNA-binding protein, was found co-localized with trypanosomatids telomeres and showed a high preference for the telomeric G-rich strand. METHODS AND RESULTS: We predicted the absence of structural homologs of OB-fold containing TEBPs in the Leishmania sp. genome using structural comparisons. We demonstrated by molecular docking that the ssDNA binding mode of LaRPA-1 shares features with the higher eukaryotes POT1 and RPA-1 crystal structures ssDNA binding mode. Using fluorescence spectroscopy, protein-DNA interaction assays, and FRET, we respectively show that LaRPA-1 shares some telomeric functions with the classical TEBPs since it can bind at least one telomeric repeat, protect the telomeric G-rich DNA from 3'-5' Exonuclease I digestion, and unfold telomeric G-quadruplex. CONCLUSIONS: Our results suggest that RPA-1 emerges as a TEBP in trypanosomatids, and in this context, we present two possible evolutionary landscapes of trypanosomatids RPA-1 that could reflect upon the evolution of OB-fold containing TEBPs from all eukaryotes.

ssDNA diffuses along replication protein A via a reptation mechanism.

Replication protein A (RPA) plays a critical role in all eukaryotic DNA processing involving single-stranded DNA (ssDNA). Contrary to the notion that RPA provides solely inert protection to transiently formed ssDNA, the RPA-ssDNA complex acts as a dynamic DNA processing unit. Here, we studied the diffusion of RPA along 60 nt ssDNA using a coarse-grained model in which the ssDNA-RPA interface was modeled by both aromatic and electrostatic interactions. Our study provides direct evidence of bulge formation during the diffusion of ssDNA along RPA. Bulges can form at a few sites along the interface and store 1-7 nt of ssDNA whose release, upon bulge dissolution, leads to propagation of ssDNA diffusion. These findings thus support the reptation mechanism, which involves bulge formation linked to the aromatic interactions, whose short range nature reduces cooperativity in ssDNA diffusion. Greater cooperativity and a larger diffusion coefficient for ssDNA diffusion along RPA are observed for RPA variants with weaker aromatic interactions and for interfaces homogenously stabilized by electrostatic interactions. ssDNA propagation in the latter instance is characterized by lower probabilities of bulge formation; thus, it may fit the sliding-without-bulge model better than the reptation model. Thus, the reptation mechanism allows ssDNA mobility despite the extensive and high affinity interface of RPA with ssDNA. The short-range aromatic interactions support bulge formation while the long-range electrostatic interactions support the release of the stored excess ssDNA in the bulge and thus the overall diffusion.

A key interaction with RPA orients XPA in NER complexes.

The XPA protein functions together with the single-stranded DNA (ssDNA) binding protein RPA as the central scaffold to ensure proper positioning of repair factors in multi-protein nucleotide excision repair (NER) machinery. We previously determined the structure of a short motif in the disordered XPA N-terminus bound to the RPA32C domain. However, a second contact between the XPA DNA-binding domain (XPA DBD) and the RPA70AB tandem ssDNA-binding domains, which is likely to influence the orientation of XPA and RPA on the damaged DNA substrate, remains poorly characterized. NMR was used to map the binding interfaces of XPA DBD and RPA70AB. Combining NMR and X-ray scattering data with comprehensive docking and refinement revealed how XPA DBD and RPA70AB orient on model NER DNA substrates. The structural model enabled design of XPA mutations that inhibit the interaction with RPA70AB. These mutations decreased activity in cell-based NER assays, demonstrating the functional importance of XPA DBD-RPA70AB interaction. Our results inform ongoing controversy about where XPA is bound within the NER bubble, provide structural insights into the molecular basis for malfunction of disease-associated XPA missense mutations, and contribute to understanding of the structure and mechanical action of the NER machinery.

Human replication protein A induces dynamic changes in single-stranded DNA and RNA structures.

Replication protein A (RPA) is the major eukaryotic ssDNA-binding protein and has essential roles in genome maintenance. RPA binds to ssDNA through multiple modes, and recent studies have suggested that the RPA-ssDNA interaction is dynamic. However, how RPA alternates between different binding modes and modifies ssDNA structures in this dynamic interaction remains unknown. Here, we used single-molecule FRET to systematically investigate the interaction between human RPA and ssDNA. We show that RPA can adopt different types of binding complexes with ssDNAs of different lengths, leading to the straightening or bending of the ssDNAs, depending on both the length and structure of the ssDNA substrate and the RPA concentration. Importantly, we noted that some of the complexes are highly dynamic, whereas others appear relatively static. On the basis of the above observations, we propose a model explaining how RPA dynamically engages with ssDNA. Of note, fluorescence anisotropy indicated that RPA can also associate with RNA but with a lower binding affinity than with ssDNA. At the single-molecule level, we observed that RPA is undergoing rapid and repetitive associations with and dissociation from the RNA. This study may provide new insights into the rich dynamics of RPA binding to ssDNA and RNA.

Development of Replication Protein A-Conjugated Gold Nanoparticles for Highly Sensitive Detection of Disease Biomarkers.

Paper-based lateral flow immunoassays (LFIAs) using conventional sandwich-type immunoassays are one of the most commonly used point-of-care (PoC) tests. However, the application of gold nanoparticles (AuNPs) in LFIAs does not meet sensitivity requirements for the detection of infectious diseases or biomarkers present at low concentrations in body fluids because of the limited number of AuNPs that can bind to the target. To overcome this problem, we first developed a single-stranded DNA binding protein (RPA70A, DNA binding domain A of human Replication Protein A 70 kDa) conjugated to AuNPs for a sandwich assay using a capture antibody immobilized in the LFIA and an aptamer as a detection probe, thus, enabling signal intensity enhancement by attaching several AuNPs per aptamer. We applied this method to detect the influenza nucleoprotein (NP) and cardiac troponin I (cTnI). We visually detected spiked targets at a low femtomolar range, with limits of detection for NP in human nasal fluid and for cTnI in serum of 0.26 and 0.23 pg·mL-1, respectively. This technique showed significantly higher sensitivity than conventional methods that are widely used in LFIAs involving antibody-conjugated AuNPs. These results suggest that the proposed method can be universally applied to the detection of substances requiring high sensitivity and can be used in the field of PoC testing for early disease diagnosis.