Meni Wanunu

Nanopores are compelling tools for ultrafast single-molecule protein analysis. Recent investigations into both enzyme-free and enzyme-regulated transport mechanisms suggest that the faster kinetics of enzyme-free transport enable higher throughput, albeit requiring speed control for accurate protein characterization. Here we investigate the signals obtained from chemically unfolded proteins when transported through wild-type cytotoxin K (CytK) in an enzyme-free manner. We designed, expressed, and measured synthetic block isoforms derived from maltose-binding protein that contain repeat sub-sequences as benchmark molecules. Surprisingly, reverse capture of proteins (i.e., from the β-barrel side) proceeds with ~2.5 orders of magnitude higher rate than forward capture, allowing high-rate protein fingerprinting at sub-nM concentrations. We utilize high-voltage reverse capture for rapid analysis of the block isoforms, and found that the ionic current signal shapes, arising from stochastic movements with 4-7 amino-acid-long steps, reproducibly follow the number and order of sequence repeats. We corroborate our results with ionic current signatures obtained from molecular dynamics simulations, exhibiting matching signature shapes. Lastly, we demonstrate an example life-science research use case through rapid nanopore quantification of leaky and overexpressed proteins that contain short electrophoretic tags directly from crude bacterial lysate in under 30 minutes, which allows for rapid and selective quantification of full-length protein expression levels.

Nanopore sequencing has enabled various layers of information about DNA and RNA sequence isoforms and chemical modifications. Yet, the archipelago of disjoint nanopore analysis tools makes navigating among these a significant challenge for the nanopore user. We present NanoCortex, a unified autonomous agentic framework designed to bridge this shortcoming by providing end-to-end data processing which ranges from raw signal basecalling to biological interpretation. Built upon Gemini API services that incur usage-based API costs and orchestrated through the Gemini Agent Development Kit (ADK), the system utilizes a multi-agent architecture to autonomously perform task parsing, code generation, iterative code-level self-correction of code, and scientific interpretation. Following code generation, the code can be used offline. Benchmarking reveals that NanoCortex achieves significantly higher usability across complex analytical tasks compared to general-purpose large language models. The framework seamlessly integrates experimental data with meta-analysis of publicly available, biological databases to facilitate the extraction of biologically meaningful insights from sequencing data without cumbersome computational steps.

Despite advances in deciphering chromatin structure and dynamics in recent years, mapping the sequence position and combinatorial modifications of individual nucleosomes, the building blocks of chromatin, has yet to be achieved. In this work, we develop SM-NucSeq: a technology that combines single-molecule immunoaffinity detection with single-molecule real-time (SMRT) sequencing in zero-mode waveguides (ZMWs), which are sub-wavelength well-like structures that confine the depth of the excitation beam to the nanometer scale. We show that SM-NucSeq can detect histone modifications on intact nucleosomes and identify their underlying DNA sequences. We leverage the ZMW chips to load nucleosome/polymerase complexes and detect co-occurring histone modifications with fluorescent antibodies, validating each step on a chromatically resolved micromirror TIRF microscope. Finally, the application of SM-NucSeq reveals that DNA synthesis rates are reduced in nucleosomes compared to the double-stranded DNA control sample and that DNA synthesis rate profiles are histone-modification-dependent.

RNA is at the forefront of therapeutics and gene editing technologies. Yet, RNA synthesis remains expensive and low-yield. Consequently, most oligo manufacturers abstain from synthesizing RNA oligos longer than 60-mers. Solid-phase synthesis is the current standard production method but is often fraught with low coupling yields for canonical nucleotides and even poorer coupling for modifications. This results in high levels of byproducts such as truncations and RNA infidelity. Existing analytical methods can only provide quality control metrics such as RNA length distribution or limited composition information for short oligos. Here, we developed a standard quality control metric using Oxford Nanopore direct RNA sequencing to obtain direct insight into RNA length distribution, sequence, and presence of RNA modification sites. Our pipeline identifies error-prone regions and truncation sites that occur during synthesis. Furthermore, problematic steps in the synthesis are identified and repaired. We show that our platform can produce and assess CRISPR guide RNAs with high-fidelity and higher cleavage activity, and further, that modifications can be reliably detected. We envision that our tool will serve as an integral method for quality control pipelines that assess the integrity and accuracy of synthetic RNAs and guide the improved synthesis and yield of synthesized RNAs. Graphical

Immortalized cell lines are commonly used as proxies for primary cells in human biology research. For example, Jurkat leukemic T cells fundamentally contributed to uncovering T-cell signaling, activation, and immune responses. However, the immortalization process can alter key cellular properties, and researchers widely believe this process could significantly change RNA modification machinery and modification sites. In this study, we focus on pseudouridine (ψ), one of the most abundant mRNA modifications, and compare ψ profiles in mRNA from primary and immortalized T cells using direct RNA sequencing (DRS). Surprisingly, 87% of ψ-sites were shared between the two cell types, primarily in transcripts encoding proteins involved in essential cellular processes, including RNA-modification regulation. Furthermore, the analysis of the 13% of sites unique to each cell type reveals that Jurkat cells contained transcripts linked to immune activation and oncogenesis, while primary T cells contained transcripts associated with calcium signaling and intracellular trafficking. We provide a list of these genes, which should be considered when using immortalized cells to study RNA modifications in immunology contexts. Most differences were driven by whether the mRNA was present or absent in the immortalized or primary cell type. Interestingly, RNA-modification enzyme expression levels were highly conserved in both cell types. This suggests that site-specific differences in ψ levels arise from regulatory processes acting in trans rather than differences in modification enzyme levels.

Nucleic acid nanoparticles (NANPs), composed of short oligonucleotides assembled into specific architectures, are emerging as a programmable platform for the regulated drug delivery of various therapeutic agents. Here, we use a nanopore "clamp" to investigate the mechanical properties of six-stranded RNA and DNA-based NANPs with the connectivity of a cube of sizes below 10 nm. When electrophoretically forced through solid-state nanopores that are smaller than the cubes, deformation of the NANPs generates prolonged electrical signatures whose durations depend on the mechanical deformability of the structures. All-atom MD simulations further reveal differences in the mechanical flexibility of DNA, RNA, modified RNA, and hybrid DNA/RNA cubes, supporting these findings at the molecular level. While DNA cubes deform and translocate through the pore, analogous RNA cubes are too stiff and cannot squeeze through at a comparable voltage, despite having the same sequence and overall shape as the DNA cubes. Further, we find that hybrid RNA/DNA cubes exhibit intermediate mechanical deformability to pure DNA or RNA cubes, indicating an additive effect of the RNA content on nanocube stiffness. Finally, different chemical modifications introduced to the strands can be used to fine-tune the mechanical properties of the NANPs.

The solid-state nanopore technique holds the potential to develop mechanically stable and miniaturized DNA sequencing devices. However, the limited temporal resolution due to the high electric field inside the nanopore and the lack of an effective speed control strategy have hindered the realization of sequencing. Here, we reported a quad-array (four nanopores milled with ∼30 nm interpore spacing as a detection unit) that induced a redistribution of the electric field inside and outside the nanopore array and offered high-resolution discrimination of four ssDNA homopolymer types. We demonstrated that the quad-nanopore array well resolved the translocation events of polyA25 and had a length resolution of 20 nt. The molecular dynamic simulation confirmed the slowed-down translocations and superior performance of a quad-nanopore array. We found that the nanopore array configuration induced a direct reduction of the electric field inside the nanopore as well as an increase in the electrical field outside the nanopore due to electric field crosstalk. This dual benefit not only reduced the large driving force on DNA but also facilitated molecule capture through nanopores, therefore decreasing the voltage thresholds. Finally, the successful discrimination of four ssDNA homopolymer types (polyA25, polyT25, polyC25, and polyG10) was achieved using a voltage as low as 30 mV with a translocation speed of 8 μs/nt. These findings provide insights into nanopore arrays for discriminating short single-stranded nucleotides with high resolution and demonstrate promising potential for developing DNA sequencers that utilize nanopore arrays as sensing units.

Immortalized cell lines are commonly used as proxies for primary cells in human biology research. For example, Jurkat leukemic T cells fundamentally contributed to uncovering T cell signaling, activation, and immune responses. However, the immortalization process can alter key cellular properties, and researchers widely believe this process could significantly change RNA modification machinery and modification sites. In this study, we focus on pseudouridine (Ψ), one of the most abundant mRNA modifications, and compare Ψ profiles in mRNA from primary and immortalized T cells using direct RNA sequencing (DRS). Surprisingly, 87% of Ψ-sites were shared between the two cell types, primarily in transcripts encoding proteins involved in essential cellular processes, including RNA-modification regulation. Furthermore, the analysis of the 13% of sites unique to each cell type reveals that Jurkat cells contained transcripts linked to immune activation and oncogenesis, while primary T cells contained transcripts associated with calcium signaling and intracellular trafficking. We provide a list of these genes, which should be considered when using immortalized cells to study RNA modifications in immunology contexts. Most differences were driven by whether the mRNA was present or absent in the immortalized or primary cell type. Interestingly, RNA-modification enzyme expression levels were highly conserved in both cell types. This suggests that site-specific differences in Ψ levels arise from regulatory processes acting in trans rather than differences in modification enzyme levels. Significance Statement: It is widely believed that RNA modification machinery and modification sites could be significantly altered in immortalized cells, yet this has never been tested. Focusing on pseudouridine (Ψ), we map Ψ in the transcriptomes of primary and immortalized human T cells. Surprisingly, most sites are conserved in the primary and immortalized T cells, with several important examples of cases with cell type specificity and should be considered on a case-by-case basis. Furthermore, we evaluated RNA-modification machinery levels in primary and immortalized T cells, finding high conservation across the cell lines. Our findings demonstrate that RNA modifications are largely conserved between primary and immortalized cells, and the edge cases can be considered individually.

Human mitochondrial tRNAs (mt-tRNAs), critical for mitochondrial biogenesis, are frequently associated with pathogenic mutations. These mt-tRNAs have unusual sequence motifs and require post-transcriptional modifications to stabilize their fragile structures. However, whether a modification that stabilizes a wild-type (WT) mt-tRNA would also stabilize its pathogenic variants is unknown. Here we show that the N¹-methylation of guanosine at position 9 (m¹G9) of mt-Leu(UAA), while stabilizing the WT tRNA, has a destabilizing effect on variants associated with MELAS (mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes). This differential effect is further demonstrated, as removal of the m¹G9 methylation, while damaging to the WT tRNA, is beneficial to the major pathogenic variant, improving the structure and activity of the variant. These results have therapeutic implications, suggesting that the N¹-methylation of mt-tRNAs at position 9 is a determinant of pathogenicity and that controlling the methylation level is an important modulator of mt-tRNA-associated diseases.

Chemical modifications to mRNA respond dynamically to environmental cues and are important modulators of gene expression. Nanopore direct RNA sequencing has been applied for assessing the presence of pseudouridine (ψ) modifications through basecalling errors and signal analysis. These approaches strongly depend on the sequence context around the modification, and the occupancies derived from these measurements are not quantitative. In this work, we combine direct RNA sequencing of synthetic RNAs bearing site-specific modifications and supervised machine learning models (ModQuant) to achieve near-analytical, site-specific ψ quantification. Our models demonstrate that the ionic current signal features important for accurate ψ classification are sequence dependent and encompass information extending beyond n + 2 and n − 2 nucleotides from the ψ site. This is contradictory to current models, which assume that accurate ψ classification can be achieved with signal information confined to the 5-nucleotide k-mer window (n + 2 and n − 2 nucleotides from the ψ site). We applied our models to quantitatively profile ψ occupancy in five mRNA sites in datasets from seven human cell lines, demonstrating conserved and variable sites. Our study motivates a wider pipeline that uses ground-truth RNA control sets with site-specific modifications for quantitative profiling of RNA modifications. The ModQuant pipeline and guide are freely available at https://github.com/wanunulab/ModQuant.