Target ssDNA Research Articles

A Novel PCR-Free Ultrasensitive GQD-Based Label-Free Electrochemical DNA Sensor for Sensitive and Rapid Detection of Francisella tularensis

Biological warfare agents are infectious microorganisms or toxins capable of harming or killing humans. Francisella tularensis is a potential bioterrorism agent that is highly infectious, even at very low doses. Biosensors for biological warfare agents are simple yet reliable point-of-care analytical tools. Developing highly sensitive, reliable, and cost-effective label-free DNA biosensors poses significant challenges, particularly when utilizing traditional techniques such as fluorescence, electrochemical methods, and others. These challenges arise primarily due to the need for labeling, enzymes, or complex modifications, which can complicate the design and implementation of biosensors. In this study, we fabricated Graphene Quantum dot (GQD)-functionalized biosensors for highly sensitive label-free DNA detection. GQDs were immobilized on the surface of screen-printed gold electrodes via mercaptoacetic acid with a thiol group. The single-stranded DNA (ssDNA) probe was also immobilized on GQDs through strong π−π interactions. The ssDNA probe can hybridize with the ssDNA target and form double-stranded DNA, leading to a decrease in the effect of GQD but a positive shift associated with the increase in DNA concentration. The specificity of the developed system was observed with different microorganism target DNAs and up to three-base mismatches in the target DNA, effectively distinguishing the target DNA. The response time for the target DNA molecule is approximately 1010 s (17 min). Experimental steps were monitored using UV/Vis spectroscopy, Atomic Force Microscopy (AFM), and electrochemical techniques to confirm the successful fabrication of the biosensor. The detection limit can reach 0.1 nM, which is two–five orders of magnitude lower than previously reported methods. The biosensor also exhibits a good linear range from 105 to 0.01 nM and has good specificity. The biosensor’s detection limit (LOD) was evaluated as 0.1 nM from the standard calibration curve, with a correlation coefficient of R2 = 0.9712, showing a good linear range and specificity. Here, we demonstrate a cost-effective, GQD-based SPGE/F. tularensis DNA test suitable for portable electrochemical devices. This application provides good perspectives for point-of-care portable electrochemical devices that integrate sample processing and detection into a single cartridge without requiring a PCR before detection. Based on these results, it can be concluded that this is the first enzyme-free electrochemical DNA biosensor developed for the rapid and sensitive detection of F. tularensis, leveraging the nanoenzyme and catalytic properties of GQDs.

Tetramerization-dependent activation of the Sir2-associated short prokaryotic Argonaute immune system

Eukaryotic Argonaute proteins (eAgos) utilize short nucleic acid guides to target complementary sequences for RNA silencing, while prokaryotic Agos (pAgos) provide immunity against invading plasmids or bacteriophages. The Sir2-domain associated short pAgo (SPARSA) immune system defends against invaders by depleting NAD+ and triggering cell death. However, the molecular mechanism underlying SPARSA activation remains unknown. Here, we present cryo-EM structures of inactive monomeric, active tetrameric and active NAD+-bound tetrameric SPARSA complexes, elucidating mechanisms underlying SPARSA assembly, guide RNA preference, target ssDNA-triggered SPARSA tetramerization, and tetrameric-dependent NADase activation. Short pAgos form heterodimers with Sir2-APAZ, favoring short guide RNA with a 5′-AU from ColE-like plasmids. RNA-guided recognition of the target ssDNA triggers SPARSA tetramerization via pAgo- and Sir2-mediated interactions. The resulting tetrameric Sir2 rearrangement aligns catalytic residue H186 for NAD+ hydrolysis. These insights advance our understanding of Sir2-domain associated pAgos immune systems and should facilitate the development of a short pAgo-associated biotechnological toolbox.

Electrochemical DNA hybridization signal amplification system using methylene blue and ascorbic acid

This study presents a method for real-time monitoring of heterogeneous DNA hybridization using chronoamperometry (CA). The electrochemical assay is based on methylene blue (MB)-labeled ssDNA target (tMB) hybridization with a complementary ssDNA capture probe (p) chemisorbed to a gold electrode through the Au-S linkage. Real-time monitoring of nucleic acid binding is enabled by introducing ascorbic acid (AA) during CA measurements at oxidative potential. After hybridization, the newly formed MB-labeled double stranded DNA (ptMB) oxidized AA and, as a result, shuttled the electrons directly to the electrode. AA-ptMB electrocatalytic cycle allows amplifying the signal from hybridized MB-labeled DNA through oxidation of AA. The process consists three particular processes: hybridization of tMB to the surface probe, reaction between AA and ptMB, and (c) the electron transfer from ptMB to the electrode. We have demonstrated that system is limited by the AA-ptMB redox reaction (bimolecular constant, k = 8 ± 0.1 M–1 s–1). AA-ptMB cycle is sequence-specific, thus fully developed current saturation curves can be used to calculate hybridization parameters and evaluate binding kinetics under various conditions. In addition, evaluating the initial hybridization rate allows quantifying the concentration of labeled ssDNA (tMB) in several minutes. This study provides the first report of a simple electrocatalytic cycle between MB-labeled DNA and a reducer AA for monitoring DNA hybridization in real time.

Rapid LAMP-driven strand displacement for PAM-free CRISPR-based pathogen diagnostics

Accurate and rapid nucleic acid testing for pathogen diagnostics is central to controlling the spread of infectious diseases. Here, we report an assay for rapid bacterial DNA detection via PAM (protospacer adjacent motif)-free Cas12f biosensor. The assay, which is named LSD12f (LAMP-driven strand displacement for Cas12f detection), involved multiplexed loop-mediated isothermal amplification (LAMP) followed by isothermal strand displacement reaction and Cas12f-mediated cleavage of a quenched fluorophore. The core of LSD12f was engineered with forward/reverse inner primers, which made LAMP products recognized and cut by enzyme digestion, triggering single-stranded DNA (ssDNA) production. Moreover, CRISPR-Cas12f could especially recognize target ssDNA region without PAM sites to reduce false-positive amplification. LSD12f could detect Streptococcus pyogenes in blood and flesh samples within 60 min with 100 % specificity and 100 % accuracy when validated by quantitative PCR (qPCR). LSD12f showed impressive specificity and sensitivity as a rapid and adaptive toolkit to broaden the use of molecular diagnostics.

Nucleic acid-functionalized nanoscale porous carbon-based electrochemical genosensor for detection of Nocardia spp. in real samples

In this study, a porous carbon derived from a metal-organic framework (PCMOF) as a target-responsive material functionalized with Nocardia particular antisense ssDNA oligonucleotide (ssDNA capture probe) was developed to construct a simple genosensor based on biogatekeeper strategy for sensitive detection of Nocardia in complex biological samples. The PCMOF with suitable pores volume was used to encapsulate electroactive dye methylene blue (MB), and the ssDNA capture probe was used as a gatekeeper to cap PCMOF. Without the presence of Nocardia target, the electrochemical signal of trapped MB was high. Upon adding the target, the hybridization of ssDNA capture probe and target led to the formation of a probe-target double-stranded (dsDNA) structure which dissociated from PCMOF and allowed MB molecules to be released. Therefore, the electrochemical signal of the genosensor decreased. The detection of Nocardia was accomplished by observing variations in the MB peak current intensity in a dose-dependent manner. For this genosensor, a linearity range from 10−18 to 10−7 M for synthetic ssDNA target and 10 to 108 copies/mL for two standard isolates, Nocardia farcinica PTCC 1309 and Nocardia brasiliensis ATCC 19296 as well as for clinical isolates (identified as Nocardia otitidiscaviarum) was observed, respectively. The detection limit (DL) values were 0.54 aM for synthetic ssDNA target and 5, 7, and 4 copies/mL for N. farcinica, N. brasiliensis, and N. otitidiscaviarum, respectively. This genosensor was also characterized by good specificity, reproducibility, and stability.

Mechanisms for HNH-mediated target DNA cleavage in type I CRISPR-Cas systems

The metagenome-derived type I-E and type I-F variant CRISPR-associated complex for antiviral defense (Cascade) complexes, fused with HNH domains, precisely cleave target DNA, representing recently identified genome editing tools. However, the underlying working mechanisms remain unknown. Here, structures of type I-FHNH and I-EHNH Cascade complexes at different states are reported. In type I-FHNH Cascade, Cas8fHNH loosely attaches to Cascade head and is adjacent to the 5' end of the target single-stranded DNA (ssDNA). Formation of the full R-loop drives the Cascade head to move outward, allowing Cas8fHNH to detach and rotate ∼150° to accommodate target ssDNA for cleavage. In type I-EHNH Cascade, Cas5eHNH domain is adjacent to the 5' end of the target ssDNA. Full crRNA-target pairing drives the lift of the Cascade head, widening the substrate channel for target ssDNA entrance. Altogether, these analyses into both complexes revealed that crRNA-guided positioning of target DNA and target DNA-induced HNH unlocking are two key factors for their site-specific cleavage of target DNA.

Trans-nuclease activity of Cas9 activated by DNA or RNA target binding.

Type V and type VI CRISPR-Cas systems have been shown to cleave nonspecific single-stranded DNA (ssDNA) or single-stranded RNA (ssRNA) in trans, but this has not been observed in type II CRISPR-Cas systems using single guide RNA. We show here that the type II CRISPR-Cas9 systems directed by CRISPR RNA and trans-activating CRISPR RNA dual RNAs show RuvC domain-dependent trans-cleavage activity for both ssDNA and ssRNA substrates. Cas9 possesses sequence preferences for trans-cleavage substrates, preferring to cleave T- or C-rich ssDNA substrates. We find that the trans-cleavage activity of Cas9 can be activated by target ssDNA, double-stranded DNA and ssRNA. The crystal structure of Cas9 in complex with guide RNA and target RNA provides a structural basis for the binding of target RNA to activate Cas9. Based on the trans-cleavage activity of Cas9 and nucleic acid amplification technology, we develop the nucleic acid detection platforms DNA-activated Cas9 detection and RNA-activated Cas9 detection, which are capable of detecting DNA and RNA samples with high sensitivity and specificity.

Development of label-free electrochemical OMP-DNA probe biosensor as a highly sensitive system to detect of citrus huanglongbing

The fabrication of the first label-free electrochemical DNA probe biosensor for highly sensitive detection of Candidatus Liberibacter asiaticus (CLas), as the causal agent of citrus huanglongbing disease, is conducted here. An OMP probe was designed based on the hybridization with its target-specific sequence in the outer membrane protein (OMP) gene of CLas. The characterization of the steps of biosensor fabrication and hybridization process between the immobilized OMP-DNA probe and the target ssDNA oligonucleotides (OMP-complementary and three mismatches OMP or OMP-mutation) was monitored using cyclic voltammetry and electrochemical impedance spectroscopy based on increasing or decreasing in the electron transfer in [Fe (CN)6]3−/4− on the modified gold electrode surface. The biosensor sensitivity indicated that the peak currents were linear over ranges from 20 to 100 nM for OMP-complementary with the detection limit of 0.026 nM (S/N = 3). The absence of any cross-interference with other biological DNA sequences confirmed a high selectivity of fabricated biosensor. Likewise, it showed good specificity in discriminating the mutation oligonucleotides from complementary target DNAs. The functional performance of optimized biosensor was achieved via the hybridization of OMP-DNA probe with extracted DNA from citrus plant infected with CLas. Therefore, fabricated biosensor indicates promise for sensitivity and early detection of citrus huanglongbing disease.

Single primer site-specific nested PCR for accurate and rapid genome-walking

Genome-walking is a molecular tool used to unveil uncharacterized DNA regions flanking a known DNA, which has been widely used in bioscience and related areas. This study developed a reliable and efficient PCR-based genome-walking approach, named as single primer site-specific nested PCR (SPN-PCR). A SPN-PCR set sequentially consists of three single-primer nested PCR amplifications. The primary relaxed thermal cycle promotes outmost nested site-specific primer (NSSP) to partially combine with numerous places on DNA template, synthesizing many single-stranded DNAs (ssDNA). Among them, the target ssDNA is exponentially amplified in the subsequent stringent cycles, as its 3′ part possesses the outmost NSSP complement; but a non-target ssDNA cannot be amplified, because it does not possess such a complement. Stringent secondary and tertiary PCRs also exclusively enrich this target DNA. Finally, the target DNA product becomes predominant. The feasibility of SPN-PCR was validated by genome-walking several selected genes from two divergent species.

A robust bioplatform based on DNA-gated nanoscale porous carbon derived from a metal-organic framework for specific detection of Herpes simplex virus type 1

In this study, a highly efficient sensing platform based on metal-organic framework-derived porous carbon (MOF-PC) and bio-gatekeeper strategy was designed for the sensitive detection of Herpes simplex virus type 1 (HSV-1). To realize the aim above, MOF-PC was obtained through the carbonization of the zeolitic imidazolate framework (ZIF-8). The electroactive dye methylene blue (MB) was then captured as a signal probe in the MOF-PC pores. Specifically, HSV-1 highly specific antisense ssDNA oligonucleotide was connected to MOF-PC through π-stacking interaction as a gatekeeper and sealed up the pores of MOF-PC. By adding the targets (synthetic ssDNA target or total genome HSV-1 DNA) and hybridization between the immobilized specific antisense ssDNA oligonucleotides and the targets, the double-stranded DNA (dsDNA) was separated from the MOF-PC surface due to the low affinity of the MOF-PC to the dsDNA. Meanwhile, MBs were released from the opened holes of MOF-PC. Therefore, the intensity of MB peak current in the absence of a target was high and then decreased by releasing and washing of MB molecules. The MB@MOF-PC responded linearly to synthetic ssDNA target in the range from 10−18 to 10−8 M and isolated total genome HSV-1 DNA in the range from 102 to 108 copies/mL. The detection limits of 0.79 aM and 83.4 copies/mL were obtained for the synthetic ssDNA target and total genome HSV-1 DNA, respectively.

Determinants of CRISPR Cas12a nuclease activation by DNA and RNA targets.

The RNA-guided CRISPR-associated (Cas) enzyme Cas12a cleaves specific double-stranded (ds-) or single-stranded (ss-) DNA targets (in cis), unleashing non-specific ssDNA cleavage (in trans). Though this trans-activity is widely coopted for diagnostics, little is known about target determinants promoting optimal enzyme performance. Using quantitative kinetics, we show formation of activated nuclease proceeds via two steps whereby rapid binding of Cas12a ribonucleoprotein to target is followed by a slower allosteric transition. Activation does not require a canonical protospacer-adjacent motif (PAM), nor is utilization of such PAMs predictive of high trans-activity. We identify several target determinants that can profoundly impact activation times, including bases within the PAM (for ds- but not ssDNA targets) and sequences within and outside those complementary to the spacer, DNA topology, target length, presence of non-specific DNA, and ribose backbone itself, uncovering previously uncharacterized cleavage of and activation by RNA targets. The results provide insight into the mechanism of Cas12a activation, with direct implications on the role of Cas12a in bacterial immunity and for Cas-based diagnostics.

A rapid VEGF-gene-sequence photoluminescence detector for osteoarthritis.

Osteoarthritis (OA) has become a serious problem to the human society for years due to its high economic burden, disability, pain, and severe impact on the patient's lifestyle. The importance of current clinical imaging modalities in the assessment of the onset and progression of OA is well recognized by clinicians, but these modalities can only detect OA in the II stage with significant structural deterioration and clinical symptoms. Blood vessel formation induced by vascular endothelial growth factor (VEGF) occurs in the early stage and throughout the entire course of OA, enables VEGF relating gene sequence to act as a biomarker in the field of early diagnosis and monitoring of the disease. Here in, a facile rapid detection of VEGF relating ssDNA sequence was developed, in which manganese-based zeolitic imidazolate framework nanoparticles (Mn-ZIF-NPs) were synthesized by a simple coprecipitation strategy, followed by the introduction and surficial absorption of probe ssDNAs and the CRISPR/Cas12a system components. Furthermore, fluorescence experiments demonstrated that the biosensor displayed a low detection limit of 2.49nM, a good linear response to the target ssDNA ranging from 10nM to 500nM, and the ability of distinguishing single nucleotide polymorphism. This finding opens a new window for the feasible and rapid detection of ssDNA molecules for the early diagnose of OA.

Detection of free DNA based on metal-enhanced fluorescence triggered by CRISPR-Cas12a and colorimetric analysis.

The CRISPR-Cas system has been found to be extremely sensitive and there is an urgent demand to extend its potential in bioassays. Herein, we developed a novel nanobiosensor to detect the human papillomavirus 16 genes (HPV-16 DNA), which is triggered by CRISPR-Cas12a to amplify the fluorescence signal by metal-enhanced fluorescence (CAMEF). Along with the changing of the fluorescence signal, the aggregation of the substrate of MEF also leads to a change in the color of the mixture solution, enabling dual signal detection with the fluorescence and the naked eye. Furthermore, the designed CAMEF probe was verified to detect the HPV-16 DNA accurately and reliably in biological samples. Triggered by the CRISPR system, the designed CAMEF probe allows quantitative detection of the HPV-16 DNA in the wide range of 10-500 pM. Owing to the MEF, the fluorescence signal of the CAMEF probe was significantly amplified with the detection limit as low as 1 pM. Besides, we can determine the concentration of HPV-16 DNA simply by the naked eye, which also drastically reduces the possibility of false-positive signals. Theoretically, the target ssDNA could be any strand of DNA obtained by designing the crRNA sequence in the CRISPR-Cas system. We believe that the designed CAMEF sensor can present a reliable approach for the accurate detection of low amounts of target ssDNA in complex biological samples.

CRISPR-Cas14a with competitive isothermal amplification for rapid visual pathogen diagnosis

The aquatic foodborne pathogen-induced infectious disease is becoming one of the killers to people’s health worldwide, attributed to its long latent period and high infectivity. It is urgently desiderated to develop simple, rapid, sensitive and universal detection techniques for early diagnosis and timely treatment of pathogens. Here, we present a novel visible CRISPR biosensing pathogen detection system named integrating competitive annealing mediated isothermal amplification (CAMP) -mediated CRISPR-Cas14a isothermal detection platform (CCIP) through CAMP with CRISPR-Cas14a. Initially, in the presence of target gene, CAMP was conducted at constant temperature to produce folded CAMP amplicon with abundant single-stranded DNA (ssDNA) target sites. When Cas14a-small guide RNA (sgRNA) binary complex specially recognized target sequences, it would trigger the cleavage collateral activity on nonspecific ssDNA fluorescence probes to produce optical signal with a quite high turnover. Furthermore, the results could be easily observed by naked eyes in an hour with high specificity and sensitivity of 102 aM. Furthermore, the method was verified with a pathogen detection result of 100% accuracy for 15 whole blood samples. Thus, CCIP is a promising point-of-care testing (POCT) technique for practical pathogen diagnosis.

Target ssDNA activates the NADase activity of prokaryotic SPARTA immune system.

Argonaute proteins (Agos), which use small RNAs or DNAs as guides to recognize complementary nucleic acid targets, mediate RNA silencing in eukaryotes. In prokaryotes, Agos are involved in immunity: the short prokaryotic Ago/TIR-APAZ (SPARTA) immune system triggers cell death by degrading NAD+ in response to invading plasmids, but its molecular mechanisms remain unknown. Here we used cryo-electron microscopy to determine the structures of inactive monomeric and active tetrameric Crenotalea thermophila SPARTA complexes, revealing mechanisms underlying SPARTA assembly, RNA-guided recognition of target single-stranded DNA (ssDNA) and subsequent SPARTA tetramerization, as well as tetramerization-dependent NADase activation. The small RNA guides Ago to recognize its ssDNA target, inducing SPARTA tetramerization via both Ago- and TIR-mediated interactions and resulting in a two-stranded, parallel, head-to-tail TIR rearrangement primed for NAD+ hydrolysis. Our findings thus identify the molecular basis for target ssDNA-mediated SPARTA activation, which will facilitate the development of SPARTA-based biotechnological tools.

Characterization and rational engineering of Butyrivibrio crossotus argonaute for improved cleavage activity

Prokaryotic Argonautes (pAgos) are becoming more popular in biotechnology due to their versatility in molecular cloning, nucleic acid detection, and gene editing. However, the increasing demand for pAgos necessitates mining or engineering more robust and efficient Agos with high cleavage activity. In this study, we focused on characterization and engineering a prokaryotic Argonaute nuclease from bacteria Butyrivibrio crossotus (BcAgo) and investigated its features, particularly its ability to process 5′-phosphorylated guides. Our findings reveal that BcAgo can efficiently employ single-stranded DNA (ssDNA) guides to slice target ssDNA, with an optimal guide interval of 15–20 nucleotides. We also discovered that mismatches within the central and 3′ supplementary regions of the guide considerably reduce BcAgo's cleavage activity. To further enhance BcAgo activity, we produced a mutant Leu678Glu using in silico docking. The mutant showed 30% and 56% improved cleavage activity at 65 °C and 37 °C, respectively, compared to the wild type. Molecular modeling studies revealed the Leu678Glu protrusion toward the catalytic pocket, and the orientation of the catalytic residue offers a structural basis for the mutant enhanced activity. These findings provide a foundation for designing pAgos with effective activity and specificity, essential for their use in biotechnological application.

Micro-interdigitated electrodes genosensor based on Au-deposited nanoparticles for early detection of cervical cancer

Genosensor-based electrodes mediated with nanoparticles (NPs) have tremendously developed in medical diagnosis. Herein, we report a facile, rapid, low cost and highly sensitive biosensing strategy for early detection of HPV 18 using gold-nanoparticles (AuNPs) deposited on micro-IDEs. This study represents surface charge transduction of micro-interdigitated electrodes (micro-IDE) alumina insulated with silica, independent and mini genosensor modified with colloidal gold NPs (AuNPs), and determination of gene hybridization for early detection of cervical cancer. The surface of AuNPs deposited micro-IDE functionalized with optimized 3-aminopropyl-triethoxysilane (APTES) followed by hybridization with deoxyribonucleic acid (DNA) virus to develop DNA genosensor. The results of ssDNA hybridization with the ssDNA target of human papillomavirus (HPV) 18 have affirmed that micro-IDE functionalized with colloidal AuNPs resulted in the lowest detection at 0.529 aM. Based on coefficient regression, micro-IDE functionalized with AuNPs produces better results in the sensitivity test (R2 = 0.99793) than unfunctionalized micro-IDE.

Unidirectional trans-cleaving behavior of CRISPR-Cas12a unlocks for an ultrasensitive assay using hybrid DNA reporters containing a 3' toehold.

CRISPR-Cas12a can induce nonspecific trans-cleavage of dsDNA substrate, including long and stable λ DNA. However, the mechanism behind this is still largely undetermined. In this study, we observed that while trans-activated Cas12a didn't cleave blunt-end dsDNA within a short reaction time, it could degrade dsDNA reporters with a short overhang. More interestingly, we discovered that the location of the overhang also affected the susceptibility of dsDNA substrate to trans-activated Cas12a. Cas12a trans-cleaved 3' overhang dsDNA substrates at least 3 times faster than 5' overhang substrates. We attributed this unique preference of overhang location to the directional trans-cleavage behavior of Cas12a, which may be governed by RuvC and Nuc domains. Utilizing this new finding, we designed a new hybrid DNA reporter as nonoptical substrate for the CRISPR-Cas12a detection platform, which sensitively detected ssDNA targets at sub picomolar level. This study not only unfolded new insight into the trans-cleavage behavior of Cas12a but also demonstrated a sensitive CRISPR-Cas12a assay by using a hybrid dsDNA reporter molecule.

Apolipoprotein E genetic analysis in unamplified genomic DNA extracts by ligase reaction and fiber optic particle plasmon resonance biosensor

Apolipoprotein E (APOE) genotyping is important for assessing the risk of late-onset Alzheimer's disease. We developed a DNA amplification-free method to perform APOE genetic analysis based on the high sensitivity of fiber optic particle plasmon resonance (FOPPR) biosensor and the specificity of ligase reaction. The method employed the dual-functional gold-iron oxide core-satellite hybrid nanoparticles (HNPs), which was modified with a single-stranded DNA (ssDNA) probe-P1, and a ssDNA probe-P2 modified with biotin to form a nanoplasmonic detection probe. Ligation of a ssDNA target complementary to the P1–P2 pair results in a ligation product with the HNP at one end and the biotin group at the other end, which can be captured by a streptavidin-functionalized sensor fiber, leading to an increase of nanoplasmonic absorption with magnitude useful for APOE genotype determination. Using synthetic ssDNA targets as standards, we achieved limits of detection in the range of 16–38 fM and the sensor responses from the mimic homozygous samples were about two times of that from mimic heterozygous samples at the same ssDNA concentration. For proof-of-principle, three genomic samples extracted from blood were analyzed and the results favorably agree with that by DNA sequencing.

Synthetic Receptor Based on a Peptide Antibiotic-Functionalized Chimera for Hybridization-Based Polynucleotide Detection.

Nanopores offer highly sensitive, low-cost, and single-molecule sensing capabilities, and the societal impact of this approach is best captured by the advent of nanopore-based DNA detection and sequencing technologies, which extract genomic information without amplification. To address a critical difficulty plaguing such undertakings involving especially protein-based nanopores isolated in lipid bilayers, namely, the formation of a stable, long-lasting single nanopore, we pioneer herein an approach for generating functional nanostructures enabling small single-stranded DNA (ssDNA) detection. We designed a dynamic hybrid construct by appending extramembrane peptide nucleic acid (PNA) segments to the C-terminus of modified ion channel-forming alamethicin monomers. We found that the resulting chimeric molecules successfully coassemble in a voltage-dependent manner in planar lipid membranes generating diameter-variable oligomers. The subsequent interaction at the flexible extramembrane segment of such formed dynamic nanopores with aqueously added complementary ssDNA fragments leads to overall conformational alterations affecting the peptide assembly state kinetics and mediated ionic current. Such recognition events were found specific to the primary structure of target ssDNA and uninhibited the presence of serum. Our platform demonstrates the feasibility of designing an entirely new class of versatile chimeric biosensors, for which, dependent upon the nature of the attached receptor moiety and underlying recognition chemistry, the applicability area may extend to other analytes.