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

Teste de biomarcador de urina sintética CRISPR-Cas para diagnóstico de câncer

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.

Autor em destaque: Ferramentas moleculares de engenharia para detecção e imagem de doenças

Teste de Biomarcador Sintético de Urina Multianalito Mediado por CRISPR-Cas para Diagnóstico Portátil

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.

Caracterização de Nanosensores e Teste de Biomarcador de Urina Mediado por CRISPR-Cas para Diagnóstico de Câncer

Para começar, dissolva dois miligramas de PEG multivalente com grupo reativo de maleimida e um mililitro de tampão fosfato 100 milimolar. Filtrar a solução através de um filtro de 0,2 micrómetros. Adicione os conjugados do peptídeo de DNA terminado com cisteína à solução.Use cromatografia de exclusão de tamanho para remover os materiais não conjugados. Execute os exemplos no PBS. Monitorar a absorbância do para detectar DNA e peptídeos.Coloque a amostra nos tubos do filtro centrífugo e concentre os nanosensores sintetizados por centrifugação de acordo com a velocidade recomendada pelo fabricante. Use uma placa de 96 poços como base para uma câmara de alojamento personalizada. Para coletar a urina para medições basais, primeiro coloque um mouse na câmara de alojamento.Aplique uma pressão suave na bexiga do rato contido para expelir qualquer urina restante para a placa. Pipetar a urina coletada em um tubo de 1,5 mililitro. Preparar 200 microlitros da solução de injeção do nanosensor em PBS estéril.Injete por via intravenosa 200 microlitros da solução em cada rato. Após uma hora da injeção, coletar as amostras de urina dos camundongos controle e experimentais. Para a detecção CRISPR baseada em fluorescência dos códigos de barras de DNA, centrifugar as amostras de urina a 800 G por cinco minutos à temperatura ambiente.Agora combine os reagentes CRISPR e, em seguida, adicione a enzima Cas12a antes de pipetar suavemente a mistura de reação para cima e para baixo. Incubar a mistura de reação a 37 graus Celsius por 30 minutos. Em seguida, execute a reação do repórter usando a mistura de DNA do repórter baseada em FRET.Adicione a mistura a uma placa de 384 poços e meça imediatamente a fluorescência a 37 graus Celsius a cada dois minutos por três horas para monitorar a cinética de clivagem. Para executar a detecção CRISPR baseada em papel, execute uma reação de repórter alternativa após a incubação da reação CRISPR usando um repórter de DNA marcado com FAM-Biotina. Depois de combinar os reagentes em uma placa de 96 poços, cubra-a com papel alumínio e, em seguida, coloque a placa em uma incubadora a 37 graus Celsius por 30 minutos.Agora adicione 80 microlitros de PBS em um poço fresco de uma placa de 96 poços. Pipetar 20 microlitros de mistura de reação do repórter neste poço. Coloque uma tira de papel de fluxo lateral em cada poço e aguarde até que o líquido atinja o topo da tira.O DNA de fita simples quimicamente modificado demonstrou ativação de Cas12a em relação ao DNA de fita dupla não modificado e ao DNA de fita simples. Moléculas de DNA modificadas na urina não processada mostraram leitura colorimétrica em tiras de papel de fluxo lateral. A banda principal da amostra indicou a liberação de FAM detectável através da clivagem do repórter desencadeada pelo DNA urinário.

Research

JoVE Journal

Bioengineering

Nanosensor Characterization and CRISPR-Cas Mediated Urine Biomarker Test for Cancer Diagnostics

CRISPR-Cas-mediated Multianalyte Synthetic Urine Biomarker Test for Portable Diagnostics

Creating synthetic biomarkers for the development of precision diagnostics has enabled detection of disease through pathways beyond those used for ...

Bioengenharia