Click Chemistry-Based DNA Labeling of Cells for Barcoding

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Click Chemistry-Based DNA Labeling of Cells for Barcoding Applications Stefan D. Gentile, Megan E. Griebel, Erik W. Anderson, and Gregory H. Underhill Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.8b00435 • Publication Date (Web): 22 Jul 2018 Downloaded from http://pubs.acs.org on July 24, 2018

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Bioconjugate Chemistry

Click Chemistry-Based DNA Labeling of Cells for Barcoding Applications Stefan D. Gentile§, Megan E. Griebel§, Erik W. Anderson§, Gregory H. Underhill§* §

University of Illinois at Urbana-Champaign, Department of Bioengineering

* Corresponding Author 2112 Everitt Laboratory 1406 West Green Street Urbana, Illinois 61801 USA (217)-244-2169 [email protected]

Abstract Cell labeling and tracking methodologies can play an important role in experiments aimed at understanding biological systems. However, many current cell labeling and tracking techniques have limitations that preclude their use in a variety of multiplexed and high throughput applications that could best represent the heterogeneity and combinatorial complexity present in physiologic contexts. Here, we demonstrate an approach for labeling, tracking, and quantifying cells using double-stranded DNA barcodes. These barcodes are introduced to the outside of the cell membrane, giving the labeled cells a unique identifier. This approach is compatible with flow cytometric and PCR-based identification and relative quantification of the presence of barcode-labeled cells. Further, utilizing this strategy, we demonstrate the capacity for sorting and enrichment of barcoded cells from a bulk population. In addition, we illustrate the design and utility of a range of orthogonal barcode sequences, which can enable the use of multiple

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independent barcodes to track, sort, and enrich multiple cell types and/or cells receiving distinct treatments from a pooled sample. Overall, this method of labeling cells has the potential to track multiple populations of cells in both high-throughput in vitro and physiologic in vivo settings. Introduction The ability to label and track cells is imperative to observing and understanding cell fate processes. There are several methods often used to both label and track cells in vivo, including but not limited to: fluorescent dyes1, 2, nanoparticles (such as quantum dots)3-5, viral constructs1, 6, 7

and metal and radiotracers6, 8. However, these techniques have limitations and drawbacks when

trying to increase throughput and multiplexing. In particular, using fluorescence to differentiate between cells of interest is limited by the number of non-overlapping independent fluorescent signals, and further, many of these methods require extensive cellular modification. The attachment of DNA oligonucleotides (oligos) to the cellular membrane has been used for almost a decade to pattern cells onto substrates and microarrays through DNA hybridization 9-18. In this process, labeled cells interact with a surface previously conjugated with a complementary DNA oligo. In addition, the DNA oligos can be used to label or image the cells by modifying the DNA backbone to incorporate or bind to a fluorescent probe. In the past, N-Hydroxysuccinimide (NHS) ester-modified DNA oligos have been used to attach to primary amine groups on the cell surface, leaving a DNA oligo anchored to the cell membrane9, 18. Other methods for attaching DNA to the cell membrane include the expression of zinc-finger proteins on the cell membrane that specifically attach to DNA oligos, the use of artificial lipids conjugated to DNA oligos to integrate directly into the membrane, or the use of DNA tagged antibodies 19-21. However, there are still several restrictions related to the applicability of these techniques. First, the sensitivity


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of detecting fluorescent DNA can be limited in some approaches. In addition, similar to traditional fluorescent probes, multiplexing capabilities can be limited when leveraging fluorescent DNA oligo-based techniques. Further, the addition of a zinc-finger probe to the cell surface requires the cell to be genetically modified prior to the initiation of the experiment, and further, there is potential cross reactivity between zinc-fingers and non-specific DNA oligos that they are not designed to capture. To mitigate some of these issues, recent efforts have begun to explore alternative methods for introducing oligos, as well as a variety of other probes, to the cell membrane. This approach is based on the modification of cell surface glycoproteins with artificial sugars that can serve as attachment points through bio-orthogonal click chemistry 10, 11, 18

. In our studies, we illustrate a straightforward method for labeling cells with DNA oligo

barcodes that is broadly compatible with both flow cytometric and PCR-based detection and quantification (Figure 1). This approach enables the independent cell labeling, and subsequent, relative cell frequency quantification in mixed populations. In addition, we demonstrate the multiplexing capabilities of this approach, as well as the sensitive detection of a small number of cells (as low as 0.01% of total) within a larger bulk population. Results and Discussion Overview of Barcoding Approach A schematic overview of the experimental methodology is provided in Figure 1. In particular, the key steps in the barcoding procedure are the following. First, cells of interest are cultured in medium containing ManNAz to introduce cell surface azides as attachment points. In parallel, a 5’ amine-modified single stranded DNA oligo (the “cell-bind” DNA or the single oligo of DNA


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that will be directly bound to the cell surface) is reacted with succinimidyl ester DIBO alkyne to introduce the cyclocotyne needed for later reaction with the cell surface azide. Next, the complementary DNA oligo is added and allowed to hybridize in a sequence-specific manner.

Figure 1:Method for Incorporating DNA Oligo Barcodes: (A) The addition of ManNAz to the cells under culturing conditions adds azides to the outside of the cell for 48 h. (B) To prepare the DNA barcodes, the cell bind DNA oligo is reacted with succinimidyl ester DIBO alkyne, and subsequently, the complement DNA oligo is added and allowed to hybridize. (C) The complete double-stranded DNA structure is introduced to the ManNAz-modified cells allowing for the reaction of the DIBO alkyne with the membrane-presented azide leading to the attachment of the oligo barcode to the cell membrane. Purple- Biotin; Green- Streptavidin, Red- Alexa-Fluor 647.


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The double stranded DNA complex is then added to azide-presenting cells to covalently attach the complex to the cell membrane surface. Here, in our presented studies, we experimentally validated and optimized these steps as well as demonstrated the utility of this barcoding approach. Further, additional methodological details are provided in Materials and Methods. Confirmation of Cell Surface Azide Addition Initially, we aimed to confirm that the ManNAz artificial sugar was sufficiently integrating into the glycoproteins containing sialic acid on the cell membrane 22-27. To verify this, we cultured A549 cells on glass coverslips in a 12 well plate for 3 days, as further outlined in the Materials and Methods. Unmodified A549 cells were cultured in parallel with the cells treated with ManNAz. Both modified and unmodified cells were labeled with DIBO Alexa-Fluor 647, fixed, and imaged using fluorescence microscopy. The unmodified cells (Figure 2A) showed no detectable Alexa-Fluor 647 signal, while the ManNAz-modified cells (Figure 2B) exhibited a strong Alexa-Fluor 647 signal. Similarly, flow cytometric analysis demonstrated that ManNAzmodified cells exhibited a substantially higher signal (approximately 3 orders of magnitude) than unmodified cells. Next, we sought to examine the duration of the detectable azide following the ManNAz treatment. ManNAz-modified cells were harvested after 2 days in the ManNAz containing medium, then replated in growth medium under standard conditions without ManNAz. Every 2 days, the cells were harvested; a subset was separated out to stain with DIBO Alexa Fluor 647 for flow cytometry, while the rest were replated under standard growth conditions. These data demonstrated that the introduced azide moieties were still detectable up to 4 days post-ManNAz treatment using flow cytometry (Figure 2C).


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Figure 2: Confirmation of the presence of cell-surface azide by ManNAz addition: (A) Unmodified cells stained with DIBO Alexa-Fluor 647 and DAPI. (B) Cells modified by adding 50 µM ManNAz to cells under standard growth conditions stained with DIBO Alexa-Fluor 647 and DAPI. (C) Cells modified with ManNAz for 48 hours (under standard conditions), passaged and replated every 48 hours. At the 48 hour increments, cells were stained with DIBO AlexaFluor 647 in solution and analyzed via flow cytometry. Azides were detectable on the cell surface 96 hours after treatment.

This process for modifying cells with ManNAz is advantageous because it can be utilized independent of cell type, and does not require any genetic modification in order to add attachment points to the surface of the cell. In addition, the azide-alkyne reaction employed is


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bio-orthogonal to natural cell processes and can be performed under aqueous and standard growth conditions23-25. Live Cell Labeling with Double-Stranded DNA After determining that the ManNAz successfully integrated into the glycoproteins in the cell membrane, we tested the capability to incorporate specific DNA barcodes through attachment to the ManNAz modified cells. A549 cells were seeded onto glass coverslips and once again treated with culture media supplemented with ManNAz. The cells were then treated with Sequence 1 double stranded DNA (dsDNA) that had previously been prepared. Following dsDNA incubation, the cells were treated with Alexa-Fluor 647 streptavidin, fixed, mounted and imaged. Fluorescence imaging assessment of cells pre-treated with ManNAz showed that the barcodes successfully attached to the azides expressed on the cells surface (Figure 3A). Barcode sequence 1 was prepared and later added to two populations of cells: untreated A549 cells and ManNAz treated A549 cells. Alexa-Fluor 647 streptavidin was introduced, for binding to the biotinylated 5’ end of sequence 1 complement, and both populations were analyzed using flow cytometry. ManNAz-modified cells demonstrated an approximately 10-fold higher amount of labeling compared to unmodified cells. The flow cytometric signal obtained for the unmodified cells tagged with dsDNA is higher than that of unmodified cells that did not receive DNA treatment, suggesting that there was some nonspecific attachment of the DNA to the cells


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Figure 3: ManNAz modified cells are successfully labeled with DNA- Double stranded DNA successfully attached to cell surface azides on modified cells using previously described methods: (A) A549 cells barcoded with biotinylated dsDNA attached to streptavidin Alexa-Fluor 647. (B) ManNAz modified cells showed close to a 10-fold increase in fluorescence compared to unmodified cells and a 1000-fold increase over control cells using flow cytometry. (C) Using our PCR based method for quantifying the number of DNA oligos attached to the cell surface, ManNAz modified cell had over 9-fold more DNA oligos than unmodified cells. *:p

Click Chemistry-Based DNA Labeling of Cells for Barcoding

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