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

CRISPR-Cas syntetisk urinbiomarkørtest til kræftdiagnostik

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 Spotlight: Tekniske molekylære værktøjer til sygdomsdetektion og billeddannelse

CRISPR-Cas-medieret multianalyt syntetisk urinbiomarkørtest til bærbar 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 og CRISPR-Cas-medieret urinbiomarkørtest til kræftdiagnostik

Til at begynde med opløses to milligram multivalent PEG med maleimid reaktiv gruppe og en milliliter 100 millimolær phosphatbuffer. Filtrer opløsningen gennem et 0,2 mikrometer filter. Tilsæt cysteinterminerede DNA-peptidkonjugater til opløsningen.Brug størrelsesekskluderingskromatografi til at fjerne de ukonjugerede materialer. Kør prøverne i PBS. Overvåg absorbansen af for at detektere DNA og peptider.Prøven anbringes i centrifugalfilterrørene, og koncentreres de syntetiserede nanosensorer ved centrifugering i henhold til producentens anbefalede hastighed. Brug en 96-brønds plade som base for et tilpasset huskammer. For at opsamle urinen til baselinemålinger skal du først placere en mus i huskammeret.Påfør let tryk på den fastholdte musens blære for at udvise eventuel resterende urin på pladen. Pipette den opsamlede urin i et 1,5 ml rør. Forbered 200 mikroliter nanosensorinjektionsopløsning i steril PBS.Injicer intravenøst 200 mikroliter af opløsningen i hver mus. Efter en times injektion indsamles urinprøverne fra kontrol- og forsøgsmusene. Til fluorescensbaseret CRISPR-detektion af DNA-stregkoderne centrifugeres urinprøverne ved 800 G i fem minutter ved stuetemperatur.Kombiner nu CRISPR-reagenserne, tilsæt derefter Cas12a-enzymet, inden du forsigtigt pipetterer reaktionsblandingen op og ned. Inkuber reaktionsblandingen ved 37 grader Celsius i 30 minutter. Kør derefter reporterreaktionen ved hjælp af den FRET-baserede reporter-DNA-blanding.Tilsæt blandingen til en plade med 384 brønde og mål straks fluorescensen ved 37 grader Celsius hvert andet minut i tre timer for at overvåge spaltningskinetikken. For at udføre papirbaseret CRISPR-detektion skal du køre en alternativ reporterreaktion efter inkubationen af CRISPR-reaktionen ved hjælp af en FAM-Biotin-mærket DNA-reporter. Efter at have kombineret reagenserne i en 96-brøndplade, skal du dække den med aluminiumsfolie og derefter placere pladen i en inkubator ved 37 grader Celsius i 30 minutter.Tilsæt nu 80 mikroliter PBS i en frisk brønd af en 96-brøndplade. Pipette 20 mikroliter reporterreaktionsblanding i denne brønd. Placer en lateral flow papirstrimmel til hver brønd, og vent, indtil væsken når toppen af strimlen.Det kemisk modificerede enkeltstrengede DNA viste Cas12a-aktivering i forhold til det umodificerede dobbeltstrengede DNA og enkeltstrengede DNA. Modificerede DNA-molekyler i uforarbejdet urin viste kolorimetrisk aflæsning på laterale flowpapirstrimler. Det førende prøvebånd indikerede frigivelsen af påviselig FAM gennem reporterspaltning udløst af urin-DNA.