Tissue-specific targeting of DNA nanodevices in a multicellular living organism
- Department of Chemistry, The University of Chicago, United States
- Grossman Institute of Neuroscience, Quantitative Biology and Human Behavior, The University of Chicago, United States
- Department of Neurobiology, The University of Chicago, United States
- Recombinant Antibody Platform (TAb-IP), Institut Curie, PSL Research University, CNRS UMR144, France
- Cell Biology and Cancer Unit, Institut Curie, PSL Research University, CNRS UMR144, France
- Department of Biological Sciences, Tata Institute of Fundamental Research, India
Figures
Figure 1
Schematic of strategies to target DNA devices to different cell types.
(a) DNA nanodevices are intrinsically targeted to coelomocytes via the endogenously expressed scavenger receptors. (b) DNA nanodevices that display a dsRNA (green) domain are targeted to intestinal …
Figure 2 with 1 supplement
DNA nanodevices are targeted to intestinal epithelial cells (IECs).
(a) Table of the composition and length of the various Alexa647 labeled DNA nanodevices used. (b) Mean Alexa647 fluorescence intensity corresponding to the uptake of the indicated nanodevice in C. …
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Figure 2—source data 1
Quantitation of uptake of nanodevices by IECs.
- https://cdn.elifesciences.org/articles/67830/elife-67830-fig2-data1-v2.xlsx
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Figure 2—source data 2
Quantitation of uptake of R100 conjugated nanodevices by IECs.
- https://cdn.elifesciences.org/articles/67830/elife-67830-fig2-data2-v2.xlsx
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Figure 2—source data 3
Quantitation of uptake of R100D50 nanodevices by IECs in various genetic backgrounds.
- https://cdn.elifesciences.org/articles/67830/elife-67830-fig2-data3-v2.xlsx
Figure 2—figure supplement 1
Uptake of nucleic acid probes in the intestinal epithelial cells.
(a) Schematic showing protocol of synthesis of azide labeled RNA for R50 and R100 strands. (b) The conjugation of Alexa 647 was confirmed by gel electrophoresis for R50. The 8% native PAGE gel was …
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Figure 2—figure supplement 1—source data 1
Pearson’s correlation coefficient (PCC) for colocalization between Rn Dn and LRO markers.
- https://cdn.elifesciences.org/articles/67830/elife-67830-fig2-figsupp1-data1-v2.xlsx
Figure 3 with 4 supplements
Identification and characterization of a sequence-specific DNA-binding recombinant antibody 9E.
(a) Schematic of phage display screen to identify DNA binders using a humanized VHH antibody library. (b) Sequence of the various dsDNA epitopes used to pinpoint the dsDNA sequence bound by the …
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Figure 3—source data 1
Binding efficiencies of the recombinant antibody 9E with dsDNA.
- https://cdn.elifesciences.org/articles/67830/elife-67830-fig3-data1-v2.xlsx
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Figure 3—source data 2
Effect of pH on binding efficiencies.
- https://cdn.elifesciences.org/articles/67830/elife-67830-fig3-data2-v2.xlsx
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Figure 3—source data 3
Relative binding constant of 9E and dsDNA.
- https://cdn.elifesciences.org/articles/67830/elife-67830-fig3-data3-v2.xlsx
Figure 3—figure supplement 1
Analysis of recombinant antibody binders of adsDNA epitope.
(a) Sequence of the biotin-labeled dsDNA epitope used for the phage display screen. (b) The epitope was immobilized via biotin (gray square) on streptavidin-conjugated magnetic beads. (c) Yield of …
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Figure 3—figure supplement 1—source data 1
Yield of the phages after each round of selection.
- https://cdn.elifesciences.org/articles/67830/elife-67830-fig3-figsupp1-data1-v2.xlsx
Figure 3—figure supplement 2
Characterization of dsDNA‐binding VHH antibodies.
(a) Schematic of the dsDNA constructs used to find the minimal dsDNA‐binding motif. All sequence details are provided in Supplementary file 1. Epitopes were immobilized on streptavidin-conjugated …
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Figure 3—figure supplement 2—source data 1
Relative binding of VHH antibodies to duplexes R1, R2, and RM.
- https://cdn.elifesciences.org/articles/67830/elife-67830-fig3-figsupp2-data1-v2.xlsx
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Figure 3—figure supplement 2—source data 2
Relative binding of VHH antibodies to duplexes R3,R4, and R5.
- https://cdn.elifesciences.org/articles/67830/elife-67830-fig3-figsupp2-data2-v2.xlsx
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Figure 3—figure supplement 2—source data 3
Relative binding of VHH antibodies to duplex R6.
- https://cdn.elifesciences.org/articles/67830/elife-67830-fig3-figsupp2-data3-v2.xlsx
Figure 3—figure supplement 3
Characterization of 9E.
(a) Purification of the dsDNA-binding 9E. Clone 9E was expressed and purified as described in Materials and methods, and the fractions obtained after elution using increasing concentrations of …
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Figure 3—figure supplement 3—source data 1
Determination of affinity of 9E for dsDNA_1.
- https://cdn.elifesciences.org/articles/67830/elife-67830-fig3-figsupp3-data1-v2.xlsx
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Figure 3—figure supplement 3—source data 2
Determination of affinity of 9E for dsDNA_2.
- https://cdn.elifesciences.org/articles/67830/elife-67830-fig3-figsupp3-data2-v2.xlsx
Figure 3—figure supplement 4
Electrophoretic mobility shift assay (EMSA) to demonstrate binding of 9E to the 4-nt minimal binding motif.
(a) Sequences, and the corresponding schematics, of DNA constructs engineered with the 4-nt epitope (red) to demonstrate binding of 9E. (b) EMSA of 9E (red crescent) to biotinylated dsDNA (lanes 1, …
Figure 4 with 2 supplements
Targeting DNA nanodevices to neurons.
(a) Schematic of the constructs used to make transgenics: (i) strategy to select transgenics based on pha-1+ worms; (ii) schematic of neuron targetable DNA nanodevice nDA647N bound to its synthetic …
Figure 4—figure supplement 1
Gel electrophoresis characterization of PCR fragements and transgenic worms.
(a, b) PCR fragments used for making various transgenic strains to target DNA probes to neurons. PCR product was confirmed by gel electrophoresis with 1% agarose gel and (c) single-worm PCR showing …
Figure 4—figure supplement 2
Representative images of transgenic worms containing both (a) rab-3p(prom1)::2xNLS::TagRFP or (b, c) unc-119::GFP or (d) prab-3::gfp::rab-3 and psnb-1::snb-1::9E injected with nDNA.
(e) Representative images of nD647 nanodevice uptake (red) in transgenic worms expressing both prab-3::gfp::rab-3 (green) and psnb-1::snb-1::9E.Also shown are normalized line intensity profiles of …
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Figure 4—figure supplement 2—source data 1
Line intensity profiles for neuron 1.
- https://cdn.elifesciences.org/articles/67830/elife-67830-fig4-figsupp2-data1-v2.xlsx
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Figure 4—figure supplement 2—source data 2
Line intensity profiles for neuron 2.
- https://cdn.elifesciences.org/articles/67830/elife-67830-fig4-figsupp2-data2-v2.xlsx
Figure 5 with 1 supplement
Mapping the pH of cell-surface versus internalized nanodevices using pHlava-9E.
(a) Representative images of pHlava-9E labeled compartments in neurons (nrn) and coelomocytes (clm) of psnb-1::snb-1::9E worms and neurons in psnb-1::odr-2::9E, acquired in the TMR (O) and ATTO 647N …
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Figure 5—source data 1
Pixel-wise line intensity profiles of pHlava-9E labeled neurons.
- https://cdn.elifesciences.org/articles/67830/elife-67830-fig5-data1-v2.xlsx
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Figure 5—source data 2
Pixel-wise line intensity profiles of pHlava-9E labeled neurons _2.
- https://cdn.elifesciences.org/articles/67830/elife-67830-fig5-data2-v2.xlsx
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Figure 5—source data 3
O/R values along line profile.
- https://cdn.elifesciences.org/articles/67830/elife-67830-fig5-data3-v2.xlsx
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Figure 5—source data 4
Distributions of O/R values for 3 representative regions.
- https://cdn.elifesciences.org/articles/67830/elife-67830-fig5-data4-v2.xlsx
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Figure 5—source data 5
pH calibration profile of pHlava-9E.
- https://cdn.elifesciences.org/articles/67830/elife-67830-fig5-data5-v2.xlsx
Figure 5—figure supplement 1
Design and characterization of pHlava-9E.
(a) Schematic showing the components of pHlava-9E. pHlava-9E is a dimeric complex comprising pH sensing strand DP, normalizing strand DA . pHlava-9E has a d(ATAA) motif at the terminus to bind 9E. (b…
Author response image 1
a) Representative images showing colocalization between LRO marker, GLO-1::GFP and R50D38 nanodevices uptaken by intestinal epithelial cells b) Pearson’s correlation coefficient (PCC) calculated of …
Additional files
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Supplementary file 1
Table listing all oligonucleotides used in this study.
- https://cdn.elifesciences.org/articles/67830/elife-67830-supp1-v2.docx
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Transparent reporting form
- https://cdn.elifesciences.org/articles/67830/elife-67830-transrepform1-v2.docx
