Structural characterization of the Plasmodium falciparum lactate transporter PfFNT alone and in complex with antimalarial compound MMV007839 reveals its inhibition mechanism.

Plasmodium falciparum, the deadliest causal agent of malaria, caused more than half of the 229 million malaria cases worldwide in 2019. The emergence and spreading of frontline drug-resistant Plasmodium strains are challenging to overcome in the battle against malaria and raise urgent demands for novel antimalarial agents. The P. falciparum formate-nitrite transporter (PfFNT) is a potential drug target due to its housekeeping role in lactate efflux during the intraerythrocytic stage. Targeting PfFNT, MMV007839 was identified as a lead compound that kills parasites at submicromolar concentrations. Here, we present 2 cryogenic-electron microscopy (cryo-EM) structures of PfFNT, one with the protein in its apo form and one with it in complex with MMV007839, both at 2.3 Å resolution. Benefiting from the high-resolution structures, our study provides the molecular basis for both the lactate transport of PfFNT and the inhibition mechanism of MMV007839, which facilitates further antimalarial drug design.

Direct single-molecule detection of CoA-SH and ATP by the membrane proteins TMEM120A and TMEM120B.

Membrane proteins are vital resources for developing biosensors. TMEM120A is a membrane protein associated with human pain transmission and lipid metabolism, and recent studies have demonstrated its ability to transport ions and bind to coenzyme A (COA-SH), indicating its potential to develop into a single-molecule sensor based on electrical methods. In this study, we investigated the ion transport properties of TMEM120A and its homolog TMEM120B on an artificial lipid bilayer using single-channel recording. The results demonstrate that both proteins can fuse into the lipid bilayer and generate stable ion currents under a bias voltage. Based on the stable ion transport capabilities of TMEM120A and TMEM120B, as well as the feature of TMEM120A binding with COA-SH, we developed these two proteins into a single-molecule sensor for detecting COA-SH and structurally similar molecules. We found that both COA-SH and ATP can reversibly bind to single TMEM120A and TMEM120B proteins embedded in the lipid bilayer and temporarily block ion currents during the binding process. By analyzing the current blocking signal, COA-SH and ATP can be identified at the single-molecule level. In conclusion, our work has provided two single-molecule biosensors for detecting COA-SH and ATP, offering insights for exploring and developing bio-inspired small molecule sensors.

Direct and Continuous Monitoring of Multicomponent Antibiotic Gentamicin in Blood at Single-Molecule Resolution.

Point-of-care monitoring of small molecules in biofluids is crucial for clinical diagnosis and treatment. However, the inherent low degree of recognition of small molecules and the complex composition of biofluids present significant obstacles for current detection technologies. Although nanopore sensing excels in the analysis of small molecules, the direct detection of small molecules in complex biofluids remains a challenge. In this study, we present a method for sensing the small molecule drug gentamicin in whole blood based on the mechanosensitive channel of small conductance in Pseudomonas aeruginosa (PaMscS) nanopore. PaMscS can directly detect gentamicin and distinguish its main components with only a monomethyl difference. The 'molecular sieve' structure of PaMscS enables the direct measurement of gentamicin in human whole blood within 10 min. Furthermore, a continuous monitoring device constructed based on PaMscS achieved continuous monitoring of gentamicin in live rats for approximately 2.5 h without blood consumption, while the drug components can be analyzed in situ. This approach enables rapid and convenient drug monitoring with single-molecule level resolution, which can significantly lower the threshold for drug concentration monitoring and promote more efficient drug use. Moreover, this work also lays the foundation for the future development of continuous monitoring technology with single-molecule level resolution in the living body.

The structure and unzipping behavior of dumbbell and hairpin DNA revealed by real-time nanopore sensing.

Hairpin structures play an essential role in DNA replication, transcription, and recombination. Single-molecule studies enable the real-time measurement and observation of the energetics and dynamics of hairpin structures, including folding and DNA-protein interactions. Nanopore sensing is emerging as a powerful tool for DNA sensing and sequencing, and previous research into hairpins using an α-hemolysin (α-HL) nanopore suggested that hairpin DNA enters from its stem side. In this work, the translocation and interaction of hairpin and dumbbell DNA samples with varying stems, loops, and toeholds were investigated systematically using a Mycobacterium smegmatis porin A (MspA) nanopore. It was found that these DNA constructs could translocate through the pore under a bias voltage above +80 mV, and blockage events with two conductance states could be observed. The events of the lower blockage were correlated with the loop size of the hairpin or dumbbell DNA (7 nt to 25 nt), which could be attributed to non-specific collisions with the pore, whereas the dwell time of events with the higher blockage were correlated with the stem length, thus indicating effective translocation. Furthermore, dumbbell DNA with and without a stem opening generated different dwell times when driven through the MspA nanopore. Finally, a new strategy based on the dwell time difference was developed to detect single nucleotide polymorphisms (SNPs). These results demonstrated that the unzipping behaviors and DNA-protein interactions of hairpin and dumbbell DNA could be revealed using nanopore technology, and this could be further developed to create sensors for the secondary structures of nucleic acids.

Ultrasensitive Nanopore Sensing of Mucin 1 and Circulating Tumor Cells in Whole Blood of Breast Cancer Patients by Analyte-Triggered Triplex-DNA Release.

The characterization of circulating tumor cells (CTCs) by liquid biopsy has a great potential for precision medicine in oncology. Here, a universal and tandem logic-based strategy is developed by combining multiple nanomaterials and nanopore sensing for the determination of mucin 1 protein (MUC1) and breast cancer CTCs in real samples. The strategy consists of analyte-triggered signal conversion, cascaded amplification via nanomaterials including copper sulfide nanoparticles (CuS NPs), silver nanoparticles (Ag NPs), and biomaterials including DNA hydrogel and DNAzyme, and single-molecule-level detection by nanopore sensing. The amplification of the non-DNA nanomaterial gives this method considerable stability, significantly lowers the limit of detection (LOD), and enhances the anti-interference performance for complicated samples. As a result, the ultrasensitive detection of MUC1 could be achieved in the range of 0.0005-0.5 pg/mL, with an LOD of 0.1 fg/mL. Moreover, we further tested MUC1 as a biomarker for the clinical diagnosis of breast cancer CTCs under double-blind conditions on the basis of this strategy, and MCF-7 cells could be accurately detected in the range from 5 to 2000 cells/mL, with an LOD of 2 cells/mL within 6 h. The detection results of the 19 clinical samples were highly consistent with those of the clinical pathological sections, nuclear magnetic resonance imaging, and color ultrasound. These results demonstrate the validity and reliability of our method and further proved the feasibility of MUC1 as a clinical diagnostic biomarker for CTCs.