Biology subjects

Johnson-Buck, A.

Publications and source records attributed to Johnson-Buck, A..

7 recordsLinked to original sources

bioRxiv2026

PINPOINT enables non-destructive nanoscopic quality control of individual lipid nanoparticles and their mRNA cargo

Quality control of lipid nanoparticles (LNPs) is central to development of mRNA therapeutics, yet ensemble measurements obscure critical heterogeneity in particle morphology and mRNA cargo loading. Here we introduce PINPOINT (Profiling Individual Nanoparticles with Precise Observation of INternalized Therapeutics), a single-particle nanoscopy platform for non-destructive, multiparametric profiling of preformulated LNP samples. Super-resolution imaging with Nile Red resolves size and morphology, while an optimized permeabilization strategy provides selective access to encapsulated mRNA without compromising LNP integrity. Single-particle fluorescence measurements, including RiboGreen and sequence-specific probes, enable quantification and validation of mRNA loading and identity. Comparative analyses of bare and encapsulated mRNA reveal kinetic and structural features, exposing heterogeneity masked by bulk assays. By integrating structural and molecular readouts at the single-particle level, PINPOINT establishes a quantitative framework for assessing of LNP composition and payload distribution, offering actionable insights for improving batch consistency and guiding the rational design of next-generation mRNA delivery systems.

biophysics↗

bioRxiv2026

FINDER converts zero-background kinetic fingerprinting into area-scalable attomolar biomarker detection

Background constrains analytical sensitivity: surveying larger sensor areas samples more analyte molecules but also accumulates false positives, limiting gains in detection performance. Here we introduce FINDER--Fluorogenic INstantaneous Digital Enumeration and Recognition--a single-molecule platform that combines kinetic fingerprinting with fluorogenic transient probes for rapid molecular classification under near-zero-background conditions. By suppressing both solution and surface-associated background at micromolar probe concentrations, FINDER classifies individual molecules within seconds-scale observation windows per field of view. This regime allows sensitivity to scale with surveyed sensor area, enabling amplification-free quantification of the miRNA cancer biomarker hsa-miR-16 with an 11 aM detection limit. FINDER further generalizes to HPV16 DNA biomarker detection, two-color RNA/DNA co-profiling, and rapid discrimination of clinically relevant EGFR single-nucleotide variants using multidimensional kinetic filtering. Rapid per-field classification permits tens of fields to be surveyed within minutes. By converting kinetic specificity into area-scalable sensitivity, FINDER enables semi-automated attomolar biomarker counting without amplification in practical workflows.

biophysics↗

bioRxiv2025

A unifying model for microRNA-guided silencing of messenger RNAs

Silencing by the miRNA-guided RNA induced silencing complex (miRISC) is dependent on Ago2-chaperoned base pairing between the miRNA 5' seed (5'S) and a complementary sequence in the 3' untranslated region of an mRNA. Prevailing mechanistic understanding posits that initial 5'S pairing can further allow functional base pair expansion into the 3' non-seed (3'NS), while functionally distinct non-canonical pairing was reported between only the 3'NS and the mRNA coding sequence. We developed single-molecule kinetics through equilibrium Poisson sampling (SiMKEPS) to measure highly precise binding and dissociation rate constants of varying-length target sequences to 5'S and 3'NS in a paradigmatic miRISC isolated from human cells, revealing distinct stable states of miRISC with mutually exclusive 5'S and 3'NS pairing. Our data suggest conformational rearrangements of the Ago2-bound miRNA that regulate alternative 5'S-and 3'NS-driven target recognition. The resulting model reconciles previously disparate observations and deepens our acumen for successfully marshaling RNA silencing therapies. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=166 SRC="FIGDIR/small/643529v1_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@1e92bb5org.highwire.dtl.DTLVardef@1348249org.highwire.dtl.DTLVardef@559fdborg.highwire.dtl.DTLVardef@1721b74_HPS_FORMAT_FIGEXP M_FIG C_FIG In brief: In this study, Chatterjee et al. developed a technique to characterize and identify multiple conformations of the RNA-induced silencing complex (RISC) present in human cells. Two distinct pathways of target recognition through different RISC conformations reconcile previously observed divergent findings on the microRNA (miRNA) binding mechanism to messenger RNA (mRNA). HighlightsO_LISiMKEPS is developed to precisely measure binding and dissociation of miRISC:mRNA C_LIO_LISingle human miRISC molecules exhibit mutually exclusive seed and non-seed pairing C_LIO_LISiMKEPS unveils conformational changes of single miRNAs bound to Ago2 C_LIO_LIResulting model reconciles previously divergent findings, supporting RNAi therapies C_LI

biophysics↗

bioRxiv2024

Mechanistic model for epigenetic maintenance by methyl-CpG-binding domain proteins

DNA methylation is a fundamental element of epigenetic regulation that is governed by the MBD protein superfamily, a group of "readers" that share a highly conserved methyl-CpG-binding domain (MBD) and mediate chromatin remodeler recruitment, transcription regulation, and coordination of DNA and histone modification. Previous work has characterized the binding affinity and sequence selectivity of MBD-containing proteins toward palindromes of 5-methylcytosine (5mC) containing 5mCpG dinucleotides, often referred to as single symmetrically methylated CpG sites. However, little is known about how MBD binding is influenced by the prototypical local clustering of methylated CpG sites and the presence of DNA structural motifs encountered, e.g., during DNA replication and transcription. Here, we use Single-Molecule Kinetics through Equilibrium Poisson Sampling (SiMKEPS) to measure precise binding and dissociation rate constants of the MBD of human protein MBD1 to DNAs with varying patterns of multiple methylated CpG sites and diverse structural motifs. MBD binding is promoted by two major properties of its DNA substrates: 1) tandem (consecutive) symmetrically methylated CpG sites in double-stranded DNA and secondary structures in single-stranded DNA; and 2) DNA forks. Based on our findings, we propose a mechanistic model for how MBD proteins contribute to epigenetic boundary maintenance between transcriptionally silenced and active genome regions.

biophysics↗

bioRxiv2024

Foundation model for efficient biological discovery in single-molecule data

Modern data-intensive techniques offer ever deeper insights into biology, but render the process of discovery increasingly complex. For example, exploiting the unique ability of single-molecule fluorescence microscopy (SMFM)1-5. to uncover rare but critical intermediates often demands manual inspection of time traces and iterative ad hoc approaches that are difficult to systematize. To facilitate systematic and efficient discovery from SMFM data, we introduce META-SiM, a transformer-based foundation model pre-trained on diverse SMFM analysis tasks. META-SiM achieves high performance--rivaling best-in-class algorithms--on a broad range of analysis tasks including trace selection, classification, segmentation, idealization, and stepwise photobleaching analysis. Additionally, the model produces high-dimensional embedding vectors that encapsulate detailed information about each trace, which the web-based META-SiM Projector (https://www.simol-projector.org) casts into lower-dimensional space for efficient whole-dataset visualization, labeling, comparison, and sharing. Combining this Projector with the objective metric of Local Shannon Entropy enables rapid identification of condition-specific behaviors, even if rare or subtle. As a result, by applying META-SiM to an existing single-molecule Forster resonance energy transfer (smFRET) dataset6, we discover a previously unobserved intermediate state in pre-mRNA splicing. META-SiM thus removes bottlenecks, improves objectivity, and both systematizes and accelerates biological discovery in complex single-molecule data.

biophysics↗

bioRxiv2024

Ultrasensitive amplification-free quantification of a methyl CpG-rich cancer biomarker by single-molecule kinetic fingerprinting

The most well-studied epigenetic marker in humans is the 5-methyl modification of cytosine in DNA, which has great potential as a disease biomarker in liquid biopsies of cell-free DNA. Currently, quantification of DNA methylation relies heavily on bisulfite conversion followed by PCR amplification and NGS or microarray analysis. PCR is subject to potential bias in differential amplification of bisulfite-converted methylated versus unmethylated sequences. Here, we combine bisulfite conversion with single-molecule kinetic fingerprinting to develop an amplification-free assay for DNA methylation at the branched-chain amino acid transaminase 1 (BCAT1) promoter. Our assay selectively responds to methylated sequences with a limit of detection below 1 fM and a specificity of 99.9999%. Evaluating complex genomic DNA matrices, we reliably distinguish 2-5% DNA methylation at the BCAT1 promoter in whole blood DNA from completely unmethylated whole-genome amplified DNA. Taken together, these results demonstrate the feasibility and sensitivity of our amplification-free, single-molecule quantification approach to improve the early detection of methylated cancer DNA biomarkers.

bioengineering↗

bioRxiv2021

Rapid Kinetic Fingerprinting of Single Nucleic Acid Molecules by a FRET-based Dynamic Nanosensor

Biofluids contain cell-free nucleic acids such as microRNAs (miRNAs) and circulating tumor-derived DNAs (ctDNAs) that have emerged as promising disease biomarkers. Conventional detection of these biomarkers by digital PCR and next generation sequencing, although highly sensitive, requires time-consuming extraction and amplification steps that increase the risk of sample loss and cross-contamination, respectively. To achieve the direct, rapid detection of miRNAs and ctDNAs with near-perfect specificity and single-molecule level sensitivity, we herein describe an accelerated amplification-free single-molecule kinetic fingerprinting. This approach, termed intramolecular single-molecule recognition through equilibrium Poisson sampling (iSiMREPS), exploits a dynamic DNA nanosensor comprising a surface anchor and a pair of fluorescent detection probes: one probe captures individual target molecules onto the surface, while the other transiently interrogates them to generate kinetic fingerprints by intramolecular single-molecule Forster resonance energy transfer (smFRET). Formamide is used to further accelerate the kinetics of probe-target interactions and fingerprinting, while background signals are reduced by removing non-target-bound probes from the surface using toehold-mediated strand displacement. We show that iSiMREPS can detect in as little as 10 seconds two distinct, promising cancer biomarkers--miR-141 and a common EGFR exon 19 deletion--reaching a limit of detection (LOD) of ~3 fM and a mutant allele fraction among excess wild-type as low as 1 in 1 million, or 0.0001%. We anticipate that iSiMREPS will find utility in research and clinical diagnostics based on its features of rapid detection, high specificity, sensitivity, and generalizability.

biophysics↗