Synthesis and characterization of peptides, oligonucleotides and peptide–oligonucleotide conjugates
All peptides and oligonucleotides were synthesized and HPLC purified by CPC Scientific and Integrated DNA Technologies (IDT), respectively. Peptide–oligonucleotide conjugates were generated by copper-free click chemistry. The conjugates were purified on an Agilent 1100 HPLC. Mass spectral analysis of the conjugates was performed on a Bruker model MicroFlex matrix-assisted laser desorption/ionization–time of flight spectrometry. Sequences of all molecules utilized are listed in Supplementary Tables 1 and 3.
Cas12a fluorescent cleavage assay
LbaCas12a (final concentration, 100 nM; New England Biolabs (NEB)) was incubated with 1× NEB Buffer 2.1, crRNA (250 nM; IDT) and complementary DNA activators (4 nM unless specifically described, in solution or spiked in urine; IDT) or urine samples collected from experimental animals, in a 50 μl reaction at 37 °C for 30 min. Reactions were diluted by a factor of 4 into 1× NEB Buffer 2.1 and ssDNA T10 F-Q reporter substrate (30 pmol; IDT) into a reaction volume of 60 μl per well and run in triplicate. LbaCas12a activation was detected at 37 °C every 2 min for 3 h by measuring fluorescence with a Tecan Infinite Pro M200 plate reader (λex = 485 nm, λem = 535 nm). Sequences of all oligonucleotides are listed in Supplementary Table 1. Fluorescence for background conditions (either no DNA activator input or no crRNA conditions) were used as negative controls. Cas12a ssDNase activity was calculated from the kinetics curve generated by the plate reader (fluorescence of the synthetic probe versus time). The initial reaction velocity (V0) corresponds to the slope of the kinetic curve’s linear phase (8–10 initial time points). Analysis was performed in Python v.3.9.
Cas12a cleavage assay with lateral-flow readout
Samples were incubated for 30 min at 37 °C as in the Cas12a activation assay described above. Reactions were then diluted by a factor of 4 into 1× NEB Buffer 2.1 and ssDNA T10 FAM–biotin reporter substrate (1 pmol; IDT) into a reaction volume of 100 µl and incubated at 37 °C for 1–3 h. HybriDetect 1 lateral-flow strips were dipped into solution (20 μl of sample with 80 μl of Milenia Hybridetect buffer). The intensity of the bands was quantified in ImageJ v.1.49.
Characterization of DNA activator concentration or length for Cas12a ssDNase activity
To identify the optimal length for detection with Cas12a, we tested truncated native and modified DNA activator lengths from 10 to 34 nt. To determine in vivo robustness, different lengths of phosphorothioate-modified DNA activators were injected at 1 nmol in BALB/c mice, and urine samples were collected 1 h after injection. Urine samples were used as DNA activators in the Cas12a fluorescent cleavage assay. Cas12a ssDNase activity triggered by each DNA activator was normalized to that of the 24-mer modified DNA activator.
Fluidigm detection and data analysis
The Cas12 detection reactions were made into two separate mixes, assay mix and sample mix, for loading onto a microfluidic Gene Expression (GE) 96.96 integrated fluidic circuit (IFC) (Fluidigm): the assay mix contained 10 μM LbaCas12a (NEB), 1× Assay Loading Reagent (Fluidigm), 1× NEB Buffer 2.1 and 1 μM crRNA for a total volume of 16 μl per reaction. The sample mix contained 25.2 U RNase inhibitor (NEB), 1× NEBuffer 2.1, 1× ROX Reference Dye (Invitrogen), 1× GE Sample Loading Reagent (Fluidigm), 9 mM MgCl2 and 500 nM quenched synthetic fluorescent DNA reporter (FAM–T10–3IABkFQ, IDT) for a total volume of 12.6 μl. Syringe and 4 μl of assay or sample mixtures were then loaded into their respective locations on a microfluidic GE 96.96 IFC and were run according to the manufacturer’s instructions. The IFC was loaded onto the Juno (Fluidigm) where the ‘Load Mix’ script was run. After proper IFC loading, images were collected over a 3 h period using a custom protocol on Biomark HD.
To analyse the data generated by the Fluidigm system, we plotted reference-normalized background-subtracted fluorescence for guide–target pairs. For a guide–target pair (at a given time point, t, and target concentration), we first computed the reference-normalized value as (median (Pt − P0)/(Rt − R0)) where Pt is the guide signal (FAM) at the time point, P0 is its background measurement before the reaction, Rt is the reference signal (ROX) at the time point, R0 is its background measurement, and the median is taken across replicates. Data was visualized using Python 3, R 4 and Prism 9.
Cloning and expression of recombinant nanobodies
Double-stranded gBlocks gene fragments encoding the nanobody of interest with flanking NcoI and BlpI restriction sites were ordered from IDT. The gene fragments were cloned into Novogen pET-28a(+) expression vector at NcoI and BlpI restriction sites and transformed into SHuffle T7 competent Escerichia coli (NEB). Bacterial colonies encoding the correct gene inserts were confirmed with Sanger sequencing. For subsequent recombinant protein production, a 500 ml secondary culture of SHuffle T7 competent E. coli. encoding the nanobody gene of interest was grown in kanamycin-supplemented LB broth at 37 °C from an overnight 3 ml primary culture until the optical density at 600 nm (OD600) reached about 0.6–0.8. Nanobody expression was then induced with an addition of isopropyl β-d-1-thiogalactopyranoside (IPTG) (0.4 mM final concentration). The culture was incubated at 27 °C for 24 h. The bacterial pellet was lysed with B-PER complete bacteria protein extraction reagent (Thermo Fisher Scientific), then purified via standard immobilized metal affinity chromatography (IMAC) with Ni-NTA agarose (Qiagen). The nanobody product was confirmed via SDS–polyacrylamide gel electrophoresis analysis. Sequences of nanobodies utilized in this study are listed in Supplementary Table 3.
Synthesis of DNA-encoded synthetic urine biomarker with a nanobody core
Nanobody (2 mg) was incubated at room temperature overnight in Pierce immobilized TCEP disulfide reducing gel (7.5% v/v) (Thermo Fisher Scientific) to selectively reduce C-terminal cysteine following a previously established protocol37. The reduced C-terminal cysteine (1 equiv.) was reacted with sulfo DBCO-maleimide crosslinker (4 equiv.) (Click Chemistry Tools) in PBS (pH 6.5, 1 mM EDTA) at room temperature for 6 h after which the excess crosslinker was removed with a disposable PD-10 desalting column (GE Healthcare Bio-Sciences). DBCO-functionalized nanobody was further refined by ÄKTA fast-protein liquid chromatography (GE Healthcare). DNA reporter conjugation was performed by incubating DBCO-functionalized nanobody (1 equiv.) with azide-functionalized DNA reporter (1.1 equiv.) in PBS (pH 7.4) at room temperature for 24 h. Excess DNA reporter was removed via size exclusion chromatography. The product was confirmed via SDS–polyacrylamide gel electrophoresis analysis and quantified with the Quant-iT OliGreen ssDNA Assay Kit. Sequences of DNA-barcoded synthetic urine biomarkers utilized in this study are listed in Supplementary Table 4.
Synthesis of DNA-encoded synthetic urine biomarkers with polymeric cores
Multivalent PEG (40 kDa, eight-arm) containing maleimide-reactive handles (JenKem Technology) was dissolved in 100 mM phosphate buffer (pH 7.0) and filtered (pore size, 0.2 μm). After filtration, the cysteine-terminated peptide–DNA conjugates were added at 2-fold molar excess to the PEG and reacted for at least 4 h at room temperature. Unconjugated molecules were separated using size-exclusion chromatography with a Superdex 200 Increase 10/300 GL column on an ÄKTA fast protein liquid chromatograph (GE Healthcare). The purified nanosensors were concentrated by spin filters (molecular weight cut-off, 10 kDa; Millipore), and quantified with a Quant-iT OliGreen ssDNA Assay Kit (Thermo Fisher Scientific). Fluorescence was read on a Tecan Infinite Pro M200 Quant-iT plate reader at λex = 485 nm, λem = 535 nm. Particles were stored at 4 °C in PBS. Dynamic light scattering (Zeta Sizer Nanoseries, Malvern Instruments) was used to characterize the hydrodynamic diameter of nanoparticles. Sequences of DNA-barcoded synthetic urine biomarkers are listed in Supplementary Table 4.
Cryogenic transmission electron microscopy
The 5-plex polymeric core-based DNA-SUBs were pooled and concentrated to 0.5 mg ml−1 by DNA concentration. Samples were loaded on a lacey copper grid coated with a continuous carbon film. The grid was then mounted on a Gatan 626 single-tilt cryoholder which was placed in the transmission electron microscope column. The samples were cooled down by liquid nitrogen and kept cold during transfer into the microscope (JEOL 2100 FEG microscope set at 200 kV; magnification, 10,000–60,000). All images were recorded using a Gatan 2kx2k UltraScan charge-coupled device camera.
Transcriptomic and proteomic analysis
RNA-Seq data of human colon adenocarcinoma were generated by the Cancer Genome Atlas Research Network (http://cancergenome.nih.gov). Differential expression analyses were carried out with DESeq2 1.10.1. Proteomic data on the composition of extracellular matrix in human colon cancers and normal colon tissues were obtained by mass spectrometry analysis of extracellular matrix components and are available from Matrisome (http://matrisomeproject.mit.edu/).
The mouse cell line MC26-LucF (carrying firefly luciferase, from Kenneth K. Tanabe Laboratory, Massachusetts General Hospital) was cultured in DMEM (Gibco) medium supplemented with 10% (v/v) fetal bovine serum (Gibco), 1% (v/v) penicillin/streptomycin (CellGro) at 37 °C and in 5% CO2. Human cell lines PC-3 (ATCC CRL-1435) were grown in RPMI1640 (Gibco) supplemented with 10% (v/v) fetal bovine serum and 1% (v/v) penicillin/streptomycin. RWPE1 (ATCC CRL-11609) cells were cultured in keratinocyte serum-free medium (Gibco) supplemented with 2.5 µg human recombinant epidermal growth factor and 25 mg bovine pituitary extract. All cell lines tested negative for mycoplasma contamination.
All animal studies were approved by the Massachusetts Institute of Technology (MIT) committee on animal care (MIT protocols 0420-023-23 and 0220-010-23). All experiments were conducted in compliance with institutional and national guidelines and supervised by the Division of Comparative Medicine (DCM) of MIT staff.
Female BALB/c (BALB/cAnNTac, 6–8 weeks of age; Taconic Biosciences), female NCr nude (CrTac:NCr-Foxn1nu, 4–5 weeks of age, Taconic Biosciences), and female and male KrasLSL−G12D/+;Trp53fl/fl C57L/B6 (KP) mice (8–16 weeks of age; gift from Tyler Jacks Laboratory, MIT) were used for experiments. Mice were maintained in the Koch Cancer Institute animal facility, with a 12 h light/12 h dark cycle (07:00–19:00), at ~18–23 °C and ~50% humidity. Autoclaved water and standard chow diet were accessible at all times. NCr nude mice were housed in immunodeficient-only rooms, in autoclaved cages and paper bedding and handled with sterile techniques. The health status of mice was checked weeklym and daily when tumours were >5 mm in diameter. Mice were euthanized according to the veterinarians’ criteria (for example, tumour size >1 cm, poor body conditions). We used a sample size of at least three mice per group. Group size is greater than or equal to five when comparing urinary barcode levels (two-sided t-test, α set at 0.05). Littermates of the same sex were randomly assigned to experimental and control groups.
To establish the CRC lung tumours, BALB/c female mice were inoculated by intravenous injection with the luciferase-expressing MC26-Fluc cell line (100,000 cells per mouse). Tumour progression was monitored weekly using an IVIS imaging system (PerkinElmer) and quantified on Living Image (PerkinElmer). To establish the PCa xenograft model, NCr nude female mice were inoculated with human PC-3 cell lines (5 million cells per flank, 2 flanks per mouse) while under isoflurane anaesthesia. Cells were prepared in 30% Corning Matrigel Membrane Matrix (Thermo Fisher Scientific) and low-serum media (Opti-MEM, Gibco). Tumours were measured weekly and experiments were conducted once flank tumours reached approximately 5 mm in length or width (~200 mm3) or 3 weeks after inoculation. Tumour volume was calculated by caliper measurement of the length and width of the flank; volume calculation followed the equation fx = (width2 × length)/2, where length is the longer segment.
To induce autochthonous lung tumours, we first generated KrasLSL−G12D/+;Trp53fl/fl C57L/B6 (KP) mice42,43 in which the activation of an oncogenic allele of KRAS is sufficient to initiate the tumorigenesis process, and additional deletion or point mutation of Trp53 substantially enhances tumour progression, leading to a more rapid development of adenocarcinomas that have features of a more advanced disease. Lung tumours were initiated by intratracheal administration of 50 μl adenovirus-SPC-Cre (2.5 × 108 plaque-forming units in Opti-MEM with 10 mM calcium chloride in female or male KP mice aged between 8 and 16 weeks) under isoflurane anaesthesia. Control cohorts consisted of age- and sex-matched mice that also underwent intratracheal administration of AdenoCre. The KP mice were maintained without further intervention to allow for tumour growth until experiments were performed.
Analysis of urinary DNA-barcode-activated Cas12a cleavage assay
ssDNAs (1 nmol), 5-plex DNA-barcoded PEG sensors (0.2 nmol each, 1 nmol by DNA barcode concentration in total) or DNA-barcoded nanobody sensors (1 nmol by DNA barcode concentration) were intravenously injected into experimental mice. Urine samples (100–200 μl per mouse) were collected at 12:00 each day, 1 h after DNA or sensor injection, and were assayed for Cas12a activation to determine the initial reaction velocity (V0). Mean normalization was performed on V0 values to account for animal-to-animal variation in urine concentration. In the Cas12a cleavage assay that utilized a fluorescent reporter, the y axis represents mean normalized V0_tumour-bearing animals/mean normalized V0_control animals. The same urine sample was then utilized to perform the Cas12a cleavage assay with LFA readout. The resulting paper strips were aligned and scanned simultaneously. Band intensity was quantified with ImageJ v.1.49v.
Biodistribution and pharmacokinetics studies
Near-infrared-dye-labelled agents were used to minimize interference from autofluorescent background in vivo. BALB/c mice were intravenously injected with Cy5-labelled modified or native DNA molecules (1 nmol) and urine samples were collected at 30 min and 1, 2, 3, 4 h after injection. Nanobody–DNA conjugates were coupled with sulfo-Cyanine7 NHS ester (Lumiprobe, 2 dye equiv. of protein), reacted overnight, purified by spin filtration and injected intravenously into PC-3 tumour-bearing nude mice. After 24 h, mice were euthanized and necropsy was performed to remove the tumours, lungs, heart, kidneys, liver and spleen. Urine, blood and organs were scanned using IVIS and Odyssey CLx imaging systems (LI-COR). Organ fluorescence was quantified by ImageStudio in the Odyssey CLx. Blood circulatory kinetics were monitored in BALB/c mice by serial blood draws at 10 min, 30 min, 2 h and 3 h after intravenous injection of Cy5-labelled DNA or PEG at 1 nmol dye per mouse. Blood for pharmacokinetics measurements was collected using tail vain bleeds. Blood was diluted in PBS with 5 mM EDTA to prevent clotting, centrifuged for 5 min at 5,000g, and fluorescent reporter concentration was quantified in 384-well plates relative to standards on the Odyssey CLx.
Histology, immunohistochemistry and immunofluorescence studies
Paraffin-embedded tissues were preserved in 4% paraformaldehyde overnight and stored in 70% ethanol prior to embedding into paraffin. Snap-frozen tissues were preserved in 2% paraformaldehyde for 2 h, stored in 30% sucrose overnight and frozen in optimum cutting temperature (OCT) compound at −80 °C. Snap-frozen lungs were processed through intratracheal injection of 50:50 OCT in PBS immediately after the animal had been euthanized by isoflurane overdose. The lungs were slowly frozen with OCT embedding in an isopentane/liquid nitrogen bath. Samples were sectioned into 6 µm slices. For immunohistochemistry studies, slides were stained with primary antibodies in accordance with the manufacturer’s instructions, followed by a Rabbit-on-Rodent HRP-Polymer used as received (Biocare Medical). For immunofluorescence studies, after blocking with 5% goat serum, 2% BSA and 0.1% Triton X-100 in PBS for 1 h, sections were stained with a primary antibody in 1% BSA in PBS overnight at 4 °C. Alexa Fluor conjugated secondary antibodies were incubated at 1 μg ml−1 in 1% BSA in PBS for 30 min at room temperature. Slides were sealed with ProLong Antifade Mountant (Thermo Fisher Scientific), and digitized and analysed using a 3D Histech P250 high-capacity slide scanner (PerkinElmer). Histological toxicity was evaluated by a veterinary pathologist who was blinded to the treatment groups. Primary antibodies and dilutions used are listed in Supplementary Table 5.
RNA extraction and real-time quantitative polymerase chain reaction
PC-3 and RWPE1 cells were cultured and collected after trypsinization. Tissue samples were collected by necropsy after mice had been euthanized and were immediately kept in RNAlater RNA Stabilization Reagent (Qiagen). RNA from cell pellets or cryoground tissue samples was extracted using an RNeasy Mini Kit (Qiagen). RNA was reverse transcribed into cDNA using Bio-Rad iScript Reverse Transcription Supermix on a Bio-Rad iCycler. Quantitative polymerase chain reaction amplification of the cDNA was measured after mixing with Taqman gene expression probes and Applied Biosystems TaqMan Fast Advanced Master Mix (Thermo Fisher Scientific) according to the manufacturer’s instructions. Quantitative polymerase chain reaction was performed on a Bio-Rad CFX96 Real Time System C1000 Thermal Cycler.
Recombinant protease substrate and tissue lysate proteolytic cleavage assays
Fluorogenic protease substrates with fluorescence (FAM) and quencher (CPQ2) were synthesized by CPC Scientific. Recombinant proteases were purchased from Enzo Life Sciences and R&D Systems. Tissue samples were homogenized in PBS and centrifuged at 4 °C for 5 min at 6,000g. Supernatant was further centrifuged at 14,000g for 25 min at 4 °C. Protein concentration was measured using a Thermo Fisher BCA Protein Assay Kit and prepared at 2 mg ml−1. Assays were performed in 384-well plates in triplicate in enzyme-specific buffer with peptides (1 µM) and proteases (40 nM for recombinant protease assay)/cell lysates (0.33 mg ml−1 for tissue lysate assay) in 30 µl at 37 °C. Fluorescence was measured at λex = 485 nm, λem = 535 nm on a Tecan Infinite 200pro microplate reader. Enzymes and buffer conditions are listed in Supplementary Table 6.
PSA enzyme-linked immunosorbent assay
Approximately 200 μl of blood was collected from the lateral saphenous vein of experimental animals and blood cells were pelleted immediately by centrifugation at 14,000g for 25 min at 4 °C. Plasma was stored at −80 °C prior to PSA quantification. PSA levels were measured using the PSA Quantikine ELISA kit according to the manufacturer’s protocols (R&D Systems).
Statistical analysis and reproducibility
Statistical analyses were conducted in GraphPad Prism 9. Data were displayed as means with s.e.m. Differences between groups were assessed using parametric and non-parametric group comparisons when appropriate with adjustment for multiple-hypothesis testing. Specifically, data groups were first tested for normality by the Kolmogorov–Smirnov normality test with the Dallal–Wilkinson–Lillie test for P value. Results were then tested for statistical significance by unpaired two-tailed t-test (parametric) for two-group comparisons and analysis of variance for multiple-group comparisons. Non-parametric analyses were conducted by unpaired two-tailed Mann–Whitney test. Sample sizes and statistical tests are specified in the figure legends. All experiments were repeated independently at least twice with similar results. Note that experiments were repeated and visualized by two independent researchers when results from representative experiments (such as histological or fluorescent micrographs) are shown.
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