Specific Hybridization Internalization Probes enable exact detection of nucleic acid sequences inside living cells or tissues. A Specific Hybridization Internalization Probe (SHIP) is an oligonucleotide designed to detect nucleic acid sequences inside living cells or tissues precisely. These probes combine the features of sequence specificity, efficient cellular uptake, and stability to enable real-time tracking or analysis of target nucleic acids. The SHIP assay allows the monitoring of antigen internalization in B cells.
The SHIP assay, developed by Liu and Johnston (2013), enables differentiation between internalized and non-internalized material in live cells. The assay utilizes a fluorescent internalization probe (FIP) containing a fluorescently labeled short 20-mer single-stranded DNA (ssDNA) coupled to a ligand of interest and a complementary ssDNA quenching probe (QP). Liu and Johnston used SHIP to investigate the internalization of transferrin (Tf) into CEM-NKR cells using anti-CD4 (CD4PE) as a plasma membrane and phenotyping stain. FIP-Cy5 (red) was attached to Tf using a strained cyclooctyne/succinimidyl ester linker (DIBO-SE). The complementary probe contained a 3’-Black Hole Quencher 2 (BHQ2). The quencher probe (QPc) is added to the assay after the material has been allowed to internalize. The QPc binds to FIP on the cell surface, quenching the fluorescence. However, the fluorescence of the internalized FIP is maintained because QPc cannot access the internalized material.
Utilizing the SHIP assay, Mann et al. (2016) showed that ~90% of the nanoparticles associated with 3T3 cells were internalized, compared to only 20% of those associated with CEM cells. Nanoparticle uptake occurred via a dynamin-dependent pathway, and the nanoparticles were trafficked to lysosomal compartments once internalized. These findings showed that SHIP can distinguish between nanoparticles on the outer cell membrane from internalized nanoparticles. Further, the assay allowed probing of the kinetics of nanoparticle internalization and the mechanisms by which cells take up the nanoparticles.
The SHIP assay allows the investigation of protein internalization kinetics, nanoparticle internalization, antibody-dependent phagocytosis, and MHCII turnover in dendritic cells using high-throughput flow cytometry studies. The SHIP assay is a simple, high-throughput method with broad applications in therapeutic delivery research.
Henandez-Perez & Mattila (2022) adopted the SHIP assay to study the trafficking of receptor/ligand complexes in a B lymphocytes and B cell receptor-mediated antigen internalization model system. This study demonstrated the potential of the SHIP assay for improved imaging of internalized receptor/ligand complexes. Also, the researchers established the compatibility of the assay with multiple imaging modalities, including live-cell imaging and super-resolution microscopy.
B lymphocytes, or B cells, as part of the adaptive immune system, initiate antibody responses against antigens. The cell’s endosomal machinery coordinates antigen processing and triggering of antigen responses. During this process, B cell receptors (BCR) recognize specific antigens internalized and directed to a specialized vesicular pathway. The antigen processing pathway enables controlled digestion of antigens into peptides. The peptides are loaded onto the major histocompatibility complex II (MHCII), and the peptide-MHCII complex is transported back to the cell surface and recognized by cognate CD4+ lymphocytes (T helper cells, TH cells).
Key Features of a SHIP assay
Sequence Specificity and Design: Design the probe to only hybridize with its complementary target sequence (DNA or RNA). The specificity is achieved by carefully optimizing the nucleotide sequence and length.
Example of the SHIP probe designed and utilized by Henandez-Perez & Mattila (2022):
ATTO 647N-5’-TCAGTTCAGGACCCTCGGCT-3’
BHQ3-3’-AGTCAAGTCCTGGGAGCCGT-5’
Labeling: SHIPs are labeled with fluorescent or radioactive tags to enable visualization or quantification. Dual-label systems, like quenchers and fluorophores, can be used for signal control.
Internalization Capability: SHIPs are often modified to facilitate cellular uptake. Modifications can include conjugation with cell-penetrating peptides (CPPs), cholesterol or other hydrophobic molecules, cationic polymers, or liposomes. Alternatively, the probe can be delivered via electroporation or microinjection.
Stability: To withstand intracellular conditions, SHIPs are often chemically modified, for example, with a phosphorothioate backbones, Bridged (BNA) or locked nucleic acids (LNA), or with 2'-O-methyl RNA.
Applications
Gene Expression Analysis: Monitoring mRNA or microRNA levels in real-time.
Molecular Diagnostics: Detecting specific pathogenic or disease-related nucleic acid sequences.
Live-cell Imaging: Visualizing the spatial and temporal dynamics of RNA or DNA.
Therapeutics: Delivery of antisense oligonucleotides for gene silencing or editing.
Example Workflow
Start with the design: Design the SHIP probe to specifically hybridize to a unique target sequence.
Follow up with labeling: Attach a fluorescent dye to the probe. Dyes like Cy3, Cy5, ATTO and Abberior STAR dyes, Alexa Fluor 488, Alexa Fluor 594, or fluoresceine isothiocyanate (FITC) can be used, as well as others. Also, for improved cell delivery the probes can be conjugated to lipids such as cholesterol PEG or cholesterol TEG to enhance cell internalization.
Henandez-Perez & Mattila (2022) labelled antibodies recognizing IgM BCR expressed in A20 B cells with an Abberior® STAR 635P or ATTO 647N-conjugated FIP to test the applicability of the SHIP system for the study of antigen trafficking. Flow cytometry and microscopy allowed monitoring of the internalization process.
Deliver the probes: Deliver the SHIP FIP probe first into cells through passive uptake or assisted delivery. After internalization of FIP, incubate with the quencher probe.
Detection: Upon hybridization with the target, a fluorescent signal is generated and monitored via flow cytometry or microscopy.
Reference
B cell memory [nature]
B-cell activation by armed helper T cells [NCBI book]
Hernández-Pérez S, Mattila PK. A specific hybridisation internalisation probe (SHIP) enables precise live-cell and super-resolution imaging of internalized cargo. Sci Rep. 2022 Jan 12;12(1):620. [PMC]
Liu H, Johnston APR. A programmable sensor to probe the internalization of proteins and nanoparticles in live cells. Angew. Chemie - Int. Ed. 2013;52(22):5744–5748. [PubMed]
Mann SK, Czuba E, Selby LI, Such GK, Johnston AP. Quantifying Nanoparticle Internalization Using a High Throughput Internalization Assay. Pharm Res. 2016 Oct;33(10):2421-32. [Springer]
Susa KJ, Seegar TC, Blacklow SC, Kruse AC. A dynamic interaction between CD19 and the tetraspanin CD81 controls B cell co-receptor trafficking. Elife. 2020 Apr 27;9:e52337. [PMC]
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