nanosensor characterization and crispr-cas mediated urine biomarker test for cancer diagnostics

CRISPR-Cas biomarkörtest för syntetisk urin för cancerdiagnostik

Despite the significant effort to identify tumor biomarkers such as shed proteins and DNA, the cancer diagnostic field has been strained by their low abundance or rapid degradation in circulation1. As a complementary strategy, bioengineered synthetic biomarkers that selectively respond to disease features to generate amplified signals represent new avenues towards accurate and accessible diagnostics2,3. To aid detection, these synthetic biomarkers harness tumor-dependent activation mechanisms such as enzymatic amplification to produce analytes with improved signal-to-noise ratio4. Herein, a class of cancer-associated enzymes, proteases, are leveraged to activate injectable nanoscale sensors to release disease reporters detectable from the biofluids such as blood or urine5,6. In light of tumor heterogeneity, developing a panel of protease-activated sensors allows for multianalyte tests that combine different protease cleavage events into a &#39;disease signature&#39; to assess cancer incidence and progression in a more specific, multiplexed manner.
Protease-activated synthetic biomarkers have been developed that comprise peptide substrates conjugated to the surface of an inert carrier7. When injected in vivo, these peptides are carried to the tumor whereupon enzymatic cleavage by tumor proteases release reporters into blood or urine for detection. Multiplexed detection with protease-activated synthetic biomarkers requires each synthetic biomarker within a cocktail to be labeled with a unique molecular barcode. To this end, various approaches have been developed, including mass barcodes and ligand-encoded reporters8,9,10. As opposed to these methods of multiplexing which may be limited to a few different signal possibilities, DNA barcoding affords many more combinations in accordance with the high complexity and heterogeneity of human disease states. To expand the multiplexity of synthetic biomarkers, the sensors are barcoded by labeling each reporter with a unique DNA sequence for detection via CRISPR-Cas nuclease to amplify the biofluidic signal ex vivo. These single-stranded DNA (ssDNA) barcodes are designed to bind to complementary CRISPR guide RNAs (crRNAs), activating the target-triggered collateral nuclease activity of CRISPR-Cas12a11. This nuclease activity can be employed to cleave a reporter DNA strand detected through fluorescence kinetics or using paper strips.
In addition to molecular amplification via proteases (in vivo) and CRISPR-Cas (ex vivo), another key design feature of protease-activated synthetic biomarkers involves harnessing nanomaterial pharmacokinetics to increase diagnostic signal concentration in biofluids10. One approach is the use of a nanoparticle carrier to increase the circulation time of surface-conjugated peptide substrates. A polyethylene glycol (PEG) dendrimer is selected as a nanocarrier with relatively small hydrodynamic diameter and multivalency to increase delivery to tumors. While small enough to promote tumor delivery, the size of the PEG carrier is larger than the ~5 nm size cut-off of the kidney glomerular filtration barrier so that only cleaved peptide substrates can be cleared into the urine, taking advantage of size filtration by the kidneys12. In this protocol, the multiple-step workflow is outlined for the synthesis and application of DNA-barcoded activity-based nanosensors in a preclinical murine model, highlighting the setup of the CRISPR-Cas-mediated, multianalyte synthetic urine biomarker test, which has been employed by this group to classify disease status in murine models of multiple cancer types13. Owing to the versatile design principle, all three functional components of the nanosensor - the nanocarrier (PEG polymer), the stimuli-responsive module (protease-activated substrate), and the biofluidic reporter (DNA barcode) - can be precisely interchanged according to application-specific needs, allowing for modularity by tailoring the target and release specificities.

Författare i fokus: Konstruera molekylära verktyg för sjukdomsdetektion och avbildning

CRISPR-Cas-medierat multianalyt syntetiskt urinbiomarkörtest för bärbar diagnostik

The Heart Lab is engineering molecular and cellular tools to precisely track and control disease biology. We use special information about diseased tissues to inform the design of responsive detection, imaging, and treatment tools for heterogeneous disorders. Biofluids present a convenient opportunity for disease detection and assessment, but these are often limited by low quantities of relevant analytes.As a result, much of the field is pursuing strategies to amplify disease associated signals to improve the sensitivity of diagnostic tests, like BCR and surface plasma on resonance. Rather than relying on endogenous disease biomarkers in circulation, our approach uses synthetic biomarkers to report disease activity when released from injected nanosensors. Our synthetic biomarkers are chemically stabilized DNA barcodes, which allow for multiple disease microenvironment signals to be detected at once, and identified using the corresponding barcode.

Nanosensorkarakterisering och CRISPR-Cas-medierat urinbiomarkörtest för cancerdiagnostik

Till att börja med, lös upp två milligram multivalent PEG med maleimidreaktiv grupp och en milliliter 100 millimolär fosfatbuffert. Filtrera lösningen genom ett 0,2 mikrometerfilter. Tillsätt de cysteinterminerade DNA-peptidkonjugaten till lösningen.Använd storleksuteslutningskromatografi för att ta bort de okonjugerade materialen. Kör exemplen i PBS. Övervaka absorbansen av för att detektera DNA och peptider.Placera provet i centrifugalfilterrören och koncentrera de syntetiserade nanosensorerna genom centrifugering enligt tillverkarens rekommenderade hastighet. Använd en 96-hålars platta som bas för en anpassad huskammare. För att samla upp urinen för baslinjemätningar, placera först en mus i huskammaren.Tryck försiktigt på den fastspända musens urinblåsa för att driva ut eventuell kvarvarande urin på plattan. Pipettera den uppsamlade urinen i ett 1,5 ml-rör. Förbered 200 mikroliter av nanosensorinjektionslösningen i steril PBS.Injicera 200 mikroliter av lösningen intravenöst i varje mus. Efter en timmes injektion samlas urinproverna från kontroll- och försöksmössen. För fluorescensbaserad CRISPR-detektion av DNA-streckkoderna, centrifugera urinproverna vid 800 G i fem minuter vid rumstemperatur.Kombinera nu CRISPR-reagenserna och tillsätt sedan Cas12a-enzymet innan du försiktigt pipetterar reaktionsblandningen upp och ner. Inkubera reaktionsblandningen vid 37 grader Celsius i 30 minuter. Kör sedan reporterreaktionen med hjälp av den FRET-baserade reporter-DNA-blandningen.Tillsätt blandningen till en platta med 384 brunnar och mät omedelbart fluorescensen vid 37 grader Celsius varannan minut i tre timmar för att övervaka klyvningens kinetik. För att utföra pappersbaserad CRISPR-detektion, kör en alternativ reporterreaktion efter inkubationen av CRISPR-reaktionen med hjälp av en FAM-Biotin-märkt DNA-reporter. Efter att ha kombinerat reagenserna till en 96-brunnsplatta, täck den med aluminiumfolie och placera sedan plattan i en inkubator vid 37 grader Celsius i 30 minuter.Tillsätt nu 80 mikroliter PBS i en ny brunn på en 96-hålsplatta. Pipettera 20 mikroliter reporterreaktionsblandning i denna brunn. Placera en pappersremsa med lateralt flöde i varje brunn och vänta tills vätskan når toppen av remsan.Det kemiskt modifierade enkelsträngade DNA:t visade Cas12a-aktivering i förhållande till det omodifierade dubbelsträngade DNA:t och enkelsträngade DNA:t. Modifierade DNA-molekyler i obehandlad urin visade kolorimetrisk avläsning på laterala flödesremsor. Det ledande provbandet indikerade frisättning av detekterbar FAM genom reporterklyvning utlöst av urin-DNA.