Biology subjects

Valera, E.

Publications and source records attributed to Valera, E..

5 recordsLinked to original sources

bioRxiv2026

Kinetic Modeling of Target-Amplification-Free CRISPR-Cas-Based Autocatalysis Reactions

CRISPR-Cas-based diagnostics utilize the Cas enzymes trans-cleavage activity to generate signal and have become popular platforms for sensitive nucleic acid detection. Recently, autocatalytic systems have been demonstrated to improve the time to response and sensitivity in some cases. However, mechanistic description of these assays is limited and optimization relies on simple trial-and-error. In this work, we present the first comprehensive kinetic model that integrates all major biochemical processes involved in these assays, including cleavage reactions, nucleic acid equilibrium kinetics, inhibition of trans-cleavage by single-stranded DNA, and degradation of single-stranded reaction components. We discuss the biochemical foundations and implementation of the ordinary differential equation model, which is built for adaptation to different reaction schemes. We use the full model to investigate the role of nucleic acid stability in assay performance for a typical nucleic acid design and show that our model demonstrates inhibition effects consistent with experimental data. We describe the reaction behavior, derive a simplified analytical model and compare its performance to the full analytical model. Finally, we demonstrate tools developed for rapid in silico optimization to guide the rational design of future target-amplification-free CRISPR-Cas-based autocatalysis assays.

bioengineering↗

bioRxiv2025

Transcranial Focused Ultrasound of the Human Subgenual Anterior Cingulate Reconfigures Resting State Connectivity: A Sham-Controlled fMRI Study

Transcranial focused ultrasound stimulation (tFUS) can modulate human brain activity, but its impact on large-scale functional connectivity (FC) in vivo remains incompletely characterized. Here we asked whether brief tFUS to the subgenual anterior cingulate cortex (sgACC) modulates its FC with other brain regions. In a sham-controlled, within-subject study of healthy adults (N = 16), we measured resting-state blood-oxygen-level dependent (BOLD) activity with functional magnetic resonance imaging (fMRI) before, during, and after five minutes of tFUS (i.e., five 20-second sonications followed by 40 second pauses). Subject-level sgACC-whole-brain FC was analyzed with a linear mixed-effects model (fixed: time, condition, timexcondition; random: subject). tFUS increased sgACC-whole brain connectivity after sonication relative to sham. A smaller, non-significant trend was observed during sonication. Baseline-controlled analyses were employed to rule out the possibility that the observed increase in FC was due to a "regression to the mean" effect. Interestingly, network-level analysis revealed a clear disassociation in the evolution of resting-state FC across the 15 minute recordings: during sham sessions, the sgACCs connectivity with the default mode network (DMN) increased, while FC with other networks was largely unchanged. On the other hand, active tFUS led to a notable increase in FC with non-DMN networks (especially the cognitive control network), while connectivity with the DMN was stable. These results provide whole-brain evidence that tFUS reconfigures network dynamics in the human brain, motivating clinical applications to disorders characterized by dysregulated brain activity.

neuroscience↗

bioRxiv2025

Amplification-Free Dual-Blocking Autocatalytic CRISPR-Cascade for Atto-Molar DNA Detection with Low Nonspecific Signal

Autocatalytic CRISPR architecture offers amplification-free nucleic acid detection by directly linking target recognition to self-reinforcing ribonucleoprotein (RNP) generation. However, spontaneous background activation remains a key barrier, because strand invasion or unwinding events can initiate unintended amplification and diminish assay specificity. Here, we introduce a dual-blocking CRISPR-Cascade design that independently cages both the guide RNA and trigger DNA, establishing an intrinsic AND gate to raise the effective kinetic barrier for unintended RNP formation. This strategy suppresses leakage by approximately 3- to 18-fold relative to single blocking configurations in full Cascade reactions, while preserving rapid detection (10 min), achieving single-copy sensitivity, and enabling quantitative detection. When paired with a competitive guide RNA decoy, the system further reduces background signals without affecting true target detection. Finally, we demonstrate robust Methicillin-resistant Staphylococcus aureus (MRSA) detection from whole blood in under 40 minutes including the sample purification and extraction. These results establish dual-blocking as a generalizable molecular gating framework for constructing leakage-resistant, amplification-free CRISPR systems suitable for rapid and decentralized diagnostics. SignificanceAmplification-free CRISPR diagnostics are often presented as simple positive feedback circuits, but most existing systems treat leakage, defined as target independent background activation that arises when blocked CRISPR components spontaneously form active ribonucleoprotein (RNP), as an unavoidable side effect rather than as a designable property. In particular, prior work has not explicitly accounted for two key sources of background signal in autocatalytic assays: Cas driven unwinding of blocked constructs and transient breathing of nucleic acid duplexes that intermittently expose trigger sites. Our study directly analyzes these leakage pathways in the context of switchable-cage-gRNA (scgRNA) and Cascade probe design and shows that blocking a single component is fundamentally vulnerable to both enzyme-driven strand invasion and equilibrium breathing. By contrast, we introduce a dual-blocking strategy in which both the guide RNA and the trigger DNA are gated. We further add a decoy guide RNA that competes for Cas12a. This multi-layer architecture demonstrates that robust amplification-free operation requires several coordinated barriers rather than a single switch, providing a new design principle for constructing self-amplifying CRISPR circuits with low background and robust signal-to-noise ratio.

molecular biology↗

bioRxiv2020

Rapid SARS-CoV-2 Detection and Classification Using Phase Imaging with Computational Specificity

Efforts to mitigate the COVID-19 crisis revealed that fast, accurate, and scalable testing is crucial for curbing the current impact and that of future pandemics. We propose an optical method for directly imaging unlabeled viral particles and using deep learning for detection and classification. An ultrasensitive interferometric method was used to image four virus types with nanoscale optical pathlength sensitivity. Pairing these data with fluorescence images for ground truth, we trained semantic segmentation models based on U-Net, a particular type of convolutional neural network. The trained network was applied to classify the viruses from the interferometric images only, containing simultaneously SARS-CoV-2, H1N1 (influenza-A), HAdV (adenovirus), and ZIKV (Zika). Remarkably, due to the nanoscale sensitivity in the input data, the neural network was able to identify SARS-CoV-2 vs. the other viruses with 96% accuracy. The inference time for each image is 60 ms, on a common graphic processing unit. This approach of directly imaging unlabeled viral particles may provide an extremely fast test, of less than a minute per patient. As the imaging instrument operates on regular glass slides, we envision this method as potentially testing on patient breath condensates. The necessary high throughput can be achieved by translating concepts from digital pathology, where a microscope can scan hundreds of slides automatically. One Sentence SummaryThis work proposes a rapid (<1 min.), label-free testing method for SARS-CoV-2 detection, using quantitative phase imaging and deep learning.

bioengineering↗

bioRxiv2020

Rapid Isothermal Amplification and Portable Detection System for SARS-CoV-2

The COVID-19 pandemic provides an urgent example where a gap exists between availability of state-of-the-art diagnostics and current needs. As assay details and primer sequences become widely known, many laboratories could perform diagnostic tests using methods such as RT-PCR or isothermal RT-LAMP amplification. A key advantage of RT-LAMP based approaches compared to RT-PCR is that RT-LAMP is known to be robust in detecting targets from unprocessed samples. In addition, RT-LAMP assays are performed at a constant temperature enabling speed, simplicity, and point-of-use testing. Here, we provide the details of an RT-LAMP isothermal assay for the detection of SARS-CoV-2 virus with performance comparable to currently approved tests using RT-PCR. We characterize the assay by introducing swabs in virus spiked synthetic nasal fluids, moving the swab to viral transport medium (VTM), and using a volume of that VTM for performing the amplification without an RNA extraction kit. The assay has a Limit-of-Detection (LOD) of 50 RNA copies/L in the VTM solution within 20 minutes, and LOD of 5000 RNA copies/L in the nasal solution. Additionally, we show the utility of this assay for real-time point-of-use testing by demonstrating detection of SARS-CoV-2 virus in less than 40 minutes using an additively manufactured cartridge and a smartphone-based reader. Finally, we explore the speed and cost advantages by comparing the required resources and workflows with RT-PCR. This work could accelerate the development and availability of SARS-CoV-2 diagnostics by proving alternatives to conventional laboratory benchtop tests. Significance StatementAn important limitation of the current assays for the detection of SARS-CoV-2 stem from their reliance on time- and labor-intensive and laboratory-based protocols for viral isolation, lysis, and removal of inhibiting materials. While RT-PCR remains the gold standard for performing clinical diagnostics to amplify the RNA sequences, there is an urgent need for alternative portable platforms that can provide rapid and accurate diagnosis, potentially at the point-of-use. Here, we present the details of an isothermal amplification-based detection of SARS-CoV-2, including the demonstration of a smartphone-based point-of-care device that can be used at the point of sample collection.

bioengineering↗