Maria D. Cabezas

Continuous electrochemical monitoring of small molecules remains difficult due to their small size and lack of enzymatic sensing strategies. Here, we develop a structure-switching aptamer molecular pendulum platform for reagentless electrochemical detection of small molecules, including melatonin and cortisol. Target binding induces a conformational change in the receptor, which increases the hydrodynamic diameter of the construct, thereby slowing electron transfer kinetics and resulting in a measurable time-resolved electrochemical current. To explore the feasibility of melatonin sensing in vivo, we validated the presence and circadian dynamics of melatonin in human interstitial fluid using mass spectrometry. We then demonstrated quantitative electrochemical detection of melatonin directly in human ISF, with signals that mirror the circadian profile observed by mass spectrometry. This work provides a foundation for continuous, minimally invasive monitoring of circadian biomarkers using electrochemical sensors.

A challenge for direct biosensing of small molecule biomarkers is transducing the binding of low-molecular weight targets with large affinity receptors into measurable signals. Molecular pendulum bioanalysis enables highly sensitive detection but necessitates that target binding induces a significant change in hydrodynamic radius of the sensor construct. Herein, we demonstrate that receptors with allosteric binding properties can be integrated with molecular pendulum sensors to enable electrochemical detection of steroid hormone analytes. Transcription factors are immobilized on gold electrodes via DNA linkers, forming monolayers that transduce binding events into measurable current changes upon application of a positive voltage. In the presence of the target, allosteric dissociation of the receptor from the linker induces a faster faradaic current response. Using this approach, we employ a steroid-responsive transcription factor to achieve progesterone detection in simulated biofluids and spiked biological samples with relevant sensitivity. Our work establishes a framework for integrating transcription factors as recognition elements for direct electrochemical biosensors, expanding their compatibility to a broader range of analytes.

Recent years have seen intense interest in the development of point-of-care nucleic acid diagnostic technologies to address the scaling limitations of laboratory-based approaches. Chief among these are combinations of isothermal amplification approaches with CRISPR-based detection and readouts of target products. Here, we contribute to the growing body of rapid, programmable point-of-care pathogen tests by developing and optimizing a one-pot NASBA-Cas13a nucleic acid detection assay. This test uses the isothermal amplification technique NASBA to amplify target viral nucleic acids, followed by the Cas13a-based detection of amplified sequences. We first demonstrate an in-house formulation of NASBA that enables the optimization of individual NASBA components. We then present design rules for NASBA primer sets and LbuCas13a guide RNAs for the fast and sensitive detection of SARS-CoV-2 viral RNA fragments, resulting in 20-200 aM sensitivity. Finally, we explore the combination of high-throughput assay condition screening with mechanistic ordinary differential equation modeling of the reaction scheme to gain a deeper understanding of the NASBA-Cas13a system. This work presents a framework for developing a mechanistic understanding of reaction performance and optimization that uses both experiments and modeling, which we anticipate will be useful in developing future nucleic acid detection technologies.

Recent years have seen intense interest in the development of point-of-care nucleic acid diagnostic technologies to address the scaling limitations of laboratory-based approaches. Chief among these are combinations of isothermal amplification approaches with CRISPR-based detection and readouts of target products. Here, we contribute to the growing body of rapid, programmable point-of-care pathogen tests by developing and optimizing a one-pot NASBA-Cas13a nucleic acid detection assay. This test uses the isothermal amplification technique NASBA to amplify target viral nucleic acids, followed by Cas13a-based detection of amplified sequences. We first demonstrate an in-house formulation of NASBA that enables optimization of individual NASBA components. We then present design rules for NASBA primer sets and LbuCas13a guide RNAs for fast and sensitive detection of SARS-CoV-2 viral RNA fragments, resulting in 20 – 200 aM sensitivity without any specialized equipment. Finally, we explore the combination of high-throughput assay condition screening with mechanistic ordinary differential equation modeling of the reaction scheme to gain a deeper understanding of the NASBA-Cas13a system. This work presents a framework for developing a mechanistic understanding of reaction performance and optimization that uses both experiments and modeling, which we anticipate will be useful in developing future nucleic acid detection technologies.

Natural products and secondary metabolites comprise an indispensable resource from living organisms that have transformed areas of medicine, agriculture, and biotechnology. Recent advances in high‐throughput DNA sequencing and computational analysis suggest that the vast majority of natural products remain undiscovered. To accelerate the natural product discovery pipeline, cell‐free metabolic engineering approaches used to develop robust catalytic networks are being repurposed to access new chemical scaffolds, and new enzymes capable of performing diverse chemistries. Such enzymes could serve as flexible biocatalytic tools to further expand the unique chemical space of natural products and secondary metabolites, and provide a more sustainable route to manufacture these molecules. Herein, we highlight select examples of natural product biosynthesis using cell‐free systems and propose how cell‐free technologies could facilitate our ability to access and modify these structures to transform synthetic and chemical biology.

In crude extract‐based cell‐free protein synthesis (CFPS), DNA templates are transcribed and translated into functional proteins. Although linear expression templates (LETs) are less laborious and expensive to generate, plasmid templates are often desired over polymerase chain reaction‐generated LETs due to increased stability and protection against exonucleases present in the extract of the reaction. Here we demonstrate that addition of a double stranded DNA‐binding protein to the CFPS reaction, termed single‐chain Cro protein (scCro), achieves terminal protection of LETs. This CroP‐LET (scCro‐based protection of LET) method effectively increases superfolder green fluorescent protein (sfGFP) expression levels from LETs in Escherichia coli CFPS reactions by sixfold. Our yields are comparable to other strategies that provide chemical and enzymatic DNA stabilization in E. coli CFPS. Notably, we also report that the CroP‐LET method successfully enhanced yields in CFPS platforms derived from nonmodel organisms. Our results show that CroP‐LET increased sfGFP yields by 18‐fold in the Vibrio natriegens CFPS platform. With the fast‐expanding applications of CFPS platforms, this method provides a practical and generalizable solution to protect linear expression DNA templates.

In crude extract-based cell-free protein synthesis (CFPS), DNA templates are transcribed and translated into functional proteins. Although linear expression templates (LETs) are less laborious and expensive to generate, plasmid templates are often desired over PCR-generated LETs due to increased stability and protection against exonucleases present in the extract of the reaction. Here we demonstrate that addition of a dsDNA-binding protein to the CFPS reaction, termed single-chain Cro protein (scCro), achieves terminal protection of LETs. This CroP-LET (sc Cro -based P rotection of LET ) method effectively increases sfGFP expression levels from LETs in Escherichia coli CFPS reactions by 6-fold. Our yields are comparable to other strategies that provide chemical and enzymatic DNA stabilization in E. coli CFPS. Notably, we also report that the CroP-LET method successfully enhanced yields in CFPS platforms derived from non-model organisms. Our results show that CroP-LET increased sfGFP yields by 18-fold in the Vibrio natriegens CFPS platform. With the fast-expanding applications of CFPS platforms, this method provides a practical and generalizable solution to protect linear expression DNA templates.

Cell-based assays are finding wider use in evaluating compounds in primary screens for drug development, yet it is still challenging to measure enzymatic activities as an end point in a cell-based assay. This paper reports a strategy that combines state-of-the-art cantilever free polymer pen lithography (PPL) with self-assembled monolayer laser desorption–ionization (SAMDI) mass spectrometry to guide cell localization and measure cellular enzymatic activities. Experiments are conducted with a 384 spot array, in which each spot is composed of ∼400 nanoarrays and each array has a 10 × 10 arrangement of 750 nm features that present extracellular matrix (ECM) proteins surrounded by an immobilized phosphopeptide. Cells attach to the individual nanoarrays, where they can be cultured and treated with small molecules, after which the media is removed and the cells are lysed. Phosphatase enzymes in the proximal lysate can then act on the immobilized phosphopeptide substrate to convert it to the dephosphorylated form. After the lysate is removed, the array is analyzed by SAMDI mass spectrometry to identify the extent of dephosphorylation and, therefore, the amount of enzyme activity in the cell. This novel approach of using nanopatterning to mediate cell adhesion and SAMDI to record enzyme activities in the proximal lysate will enable a broad range of cellular assays for applications in drug discovery and research not possible with conventional strategies.

Patterning nanoscale features across macroscopic areas is challenging due to the vast range of length scales that must be addressed. With polymer pen lithography, arrays of thousands of elastomeric pyramidal pens can be used to write features across centimeter-scales, but deformation of the soft pens limits resolution and minimum feature pitch, especially with polymeric inks. Here, we show that by coating polymer pen arrays with a ∼175 nm silica layer, the resulting hard transparent arrays exhibit a force-independent contact area that improves their patterning capability by reducing the minimum feature size (∼40 nm), minimum feature pitch (2 area were written using a four hundred thousand pyramid pen array. Furthermore, a new method is demonstrated for patterning macroscopic feature size gradients that vary in feature diameter by a factor of 4. Ultimately, this form of polymer pen lithography allows for patterning with the resolution of dip-pen nanolithography across centimeter scales using simple and inexpensive pen arrays. The high resolution and density afforded by this technique position it as a broad-based discovery tool for the field of nanocombinatorics.