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

CRISPR-Cas syntetisk urin biomarkørtest for kreftdiagnostikk

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.

Forfatter i søkelyset: Tekniske molekylære verktøy for sykdomsdeteksjon og bildediagnostikk

CRISPR-Cas-mediert multianalytt syntetisk urinbiomarkørtest for bærbar diagnostikk

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 og CRISPR-Cas-mediert urinbiomarkørtest for kreftdiagnostikk

For å begynne, oppløs to milligram multivalent PEG med maleimidreaktiv gruppe og en milliliter 100 millimolar fosfatbuffer. Filtrer løsningen gjennom et 0,2 mikrometer filter. Tilsett cysteinterminerte DNA-peptidkonjugater til løsningen.Bruk størrelsesekskluderingskromatografi for å fjerne de ukonjugerte materialene. Kjør prøvene i PBS. Overvåk absorbansen av for å oppdage DNA og peptider.Plasser prøven i sentrifugalfilterrørene og konsentrer de syntetiserte nanosensorene ved sentrifugering i henhold til produsentens anbefalte hastighet. Bruk en 96-brønns plate som base for et tilpasset huskammer. For å samle urinen for baseline målinger, først plassere en mus i huset kammeret.Påfør forsiktig trykk på den beherskede musens blære for å utvise gjenværende urin på platen. Pipett den oppsamlede urinen inn i et 1,5 milliliter rør. Forbered 200 mikroliter av nanosensorinjeksjonsløsningen i steril PBS.Injiser intravenøst 200 mikroliter av løsningen i hver mus. Etter en times injeksjon, samle urinprøver fra kontroll- og eksperimentmusene. For fluorescensbasert CRISPR-deteksjon av DNA-strekkodene, sentrifuger urinprøvene ved 800 G i fem minutter ved romtemperatur.Kombiner nå CRISPR-reagensene, tilsett deretter Cas12a-enzymet før du forsiktig pipetterer reaksjonsblandingen opp og ned. Inkuber reaksjonsblandingen ved 37 grader Celsius i 30 minutter. Kjør deretter reporterreaksjonen ved hjelp av den FRET-baserte reporterens DNA-blanding.Tilsett blandingen til en 384-brønnplate og mål umiddelbart fluorescensen ved 37 grader Celsius hvert annet minutt i tre timer for å overvåke spaltningskinetikken. For å utføre papirbasert CRISPR-deteksjon, kjør en alternativ reporterreaksjon etter inkubasjonen av CRISPR-reaksjonen ved hjelp av en FAM-Biotin-merket DNA-reporter. Etter å ha kombinert reagensene i en 96-brønnsplate, dekk den med aluminiumsfolie, og legg deretter platen i en inkubator ved 37 grader Celsius i 30 minutter.Legg nå 80 mikroliter PBS i en frisk brønn med en 96-brønnplate. Pipette 20 mikroliter reporterreaksjonsblanding inn i denne brønnen. Legg en lateral flow papirstrimmel til hver brønn, og vent til væsken når toppen av stripen.Det kjemisk modifiserte enkeltstrengede DNA demonstrerte Cas12a-aktivering i forhold til det umodifiserte dobbeltstrengede DNA og enkeltstrengede DNA. Modifiserte DNA-molekyler i ubehandlet urin viste kolorimetrisk avlesning på laterale strømningspapirstrimler. Det ledende prøvebåndet indikerte frigjøring av detekterbar FAM gjennom reporterspaltning utløst av urin-DNA.