two dna nanomachines map ph changes

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  • Two DNA nanomachines map pH changesalong intersecting endocytic pathways insidethe same cellSouvik Modi1, Clement Nizak2, Sunaina Surana1, Saheli Halder1 and Yamuna Krishnan1*

    DNA is a versatile scaffold for molecular sensing in living cells, and various cellular applications of DNA nanodevices havebeen demonstrated. However, the simultaneous use of different DNA nanodevices within the same living cell remains achallenge. Here, we show that two distinct DNA nanomachines can be used simultaneously to map pH gradients along twodifferent but intersecting cellular entry pathways. The two nanomachines, which are molecularly programmed to entercells via different pathways, can map pH changes within well-defined subcellular environments along both pathways insidethe same cell. We applied these nanomachines to probe the pH of early endosomes and the trans-Golgi network, in realtime. When delivered either sequentially or simultaneously, both nanomachines localized into and independently capturedthe pH of the organelles for which they were designed. The successful functioning of DNA nanodevices within livingsystems has important implications for sensing and therapies in a diverse range of contexts.

    DNA has been molecularly chiselled to create a variety of intri-cate architectures on the nanoscale1. DNA nanoarchitecturesthat can be triggered chemically or physically to switch

    between defined states (referred to as DNA nanomachines) havegreat potential for robotic and sensing applications on the nano-scale2,3, and subcellular compartments provide nanoscale environ-ments that offer rich possibilities for demonstrating thefunctionality of these nanoarchitectures4. It was recently demon-strated that a DNA nanomachine can function as a pH sensorinside endosomes in cellulo5 as well as in vivo6. Since then, a fewexciting applications of specific DNA nanostructures have beendemonstrated in cellulo69. However, achieving simultaneous func-tionality of multiple DNA nanomachines within the same cellremains a challenge, which, if realized, would open up possibilitiesof multiplexing DNA nanodevices in living systems. The precisepositioning of more than one DNA nanodevice within a subcellularenvironment and demonstration of their simultaneous functionalitytherein is therefore essential. Here, we apply the technology we termSimpHony (simultaneous pH mapping technology) to deploy twodifferently programmed DNA nanomachines along different cellu-lar endocytic pathways and map pH changes along both pathwayssimultaneously, within the same living cell.

    Programming strategy to achieve SimpHonySimultaneous mapping of an analyte such as pH using fluorescenceresonance energy transfer (FRET)-based DNA nanomachinesrequires molecular programming strategies that position each nano-machine along a distinct pathway, as well as a combination of FRETpairs with minimal crosstalk. SimpHony also requires two pH-responsive DNA nanomachines with pH-sensitive regimes suitedto the lumenal pH of the relevant intracellular organelles beinginvestigated. We chose two well-characterized pathways: the furinretrograde endocytic pathway and the transferrin endocytic/recycling pathway. Furin resides predominantly in the trans-Golginetwork (TGN), with a small steady-state population at theplasma membrane, and is transported retrogradely via the early

    endosomes/sorting endosomes (EE/SEs) and late endosomes(LEs) to the TGN in a Rab-9-dependent manner10,11. Transferrinbinds the transferrin receptor at the plasma membrane, reachesthe EE/SEs and then the perinuclear recycling endosome (RE)12.The transferrin and furin pathways merge in the EE and thensegregate thereafter into the RE and LE, respectively.

    To track the furin pathway, we used a molecularly programmedDNA nanomachine called IFu (Fig. 1a). This adopts the sameworking principle as described previously5,6 and is programmedsuch that (i) the position of the acceptor is optimized to maximizethe observed FRET, (ii) the FRET pair uses Alexa 546 as a donorfluorophore, which allows compatibility of IFu pH measurementsin the presence of ITf fluorophores, and (iii) IFu incorporates an8 bp dsDNA sequence (shaded in grey in Fig. 1a) that functionsas a binding site for an engineered protein (grey cylinders) thatenables its localization to a given endocytic pathway, namely thefurin pathway. Because furin is a retrogradely transported mem-brane protein that lacks a natural ligand, we developed a methodto specifically attach and transport a DNA nanostructure to sucha trafficking protein. This uses a sequence-specific double-strandedDNA (dsDNA) binding protein (single-chain variable fragmentrecombinant antibody, or scFv)13, which is expressed as a chimerawith furin. This sequence-specific, dsDNA-binding protein wasobtained from a phage display screen of scFv recombinant anti-bodies against a dsDNA epitope. This scFv specifically binds adsDNA sequence d(AT)4 (Fig. 1c) with a dissociation constant KDof 80 nM (Fig. 1d). Thus an N-terminal fusion of the scFvdomain with furin, when expressed inside cells, will act as an artifi-cial receptor for a DNA nanostructure that incorporates a d(AT)4sequence and will traffic the latter along the retrograde furin endo-cytic pathway into the TGN (Fig. 1b).

    To track the transferrin pathway, we engineered a two-strandedpH-sensitive DNA nanomachine, ITf (Fig. 1a). ITf uses intrastrandi-motif formation to undergo a pH-dependent conformationalchange and therefore functions as a FRET-based pH sensor.ITf has a pH-responsive C-rich segment, shown in purple in

    1National Centre for Biological Sciences, TIFR, GKVK, Bellary Road, Bangalore 560065, India, 2Laboratoire Interdisciplinaire de Physique, UMR5588CNRS-Universite Grenoble I, Grenoble, France. *e-mail: [email protected]

    ARTICLESPUBLISHED ONLINE: 26 MAY 2013 | DOI: 10.1038/NNANO.2013.92

    NATURE NANOTECHNOLOGY | VOL 8 | JUNE 2013 | www.nature.com/naturenanotechnology 459

    2013 Macmillan Publishers Limited. All rights reserved.

    mailto:[email protected]://www.nature.com/doifinder/10.1038/nnano.2013.92www.nature.com/naturenanotechnology

  • Fig. 1a, which partially base-pairs with a G-rich overhang in thecomplementary strand to form a weak duplex. At low pH, thisduplex frays, allowing the C-rich strand to fold into an intramolecu-lar i-motif. By chemically conjugating transferrin (Tf) to one of thestrands in ITf to give TfITf, one can molecularly program ITf to beconfined along the transferrin receptor pathway (Fig. 1b).

    Different cellular organelles have different lumenal pHvaluesranging from pH 4.8 (lysosome) to pH 7.1 (endoplasmicreticulum)so it is important to have DNA nanomachines witha suitable pH response for the relevant organelle as well as appro-priate FRET pairs for simultaneous measurements1416. The per-formances and working pH regimes of the DNA nanomachinesengineered for the furin and transferrin pathways, respectively,are shown in Fig. 1e,f. From the many fluorophore pairs tried,we found the best FRET pairs to be Alexa 488/Alexa 647(A488/A647) and Alexa 546/Alexa 647 (A546/A647), whichdemonstrated maximal FRET and minimal crosstalk when usedsimultaneously. Thus, ITf carried A488/A647 as its FRET pair(ITfA488/A647) and I

    Fu carried A546/A647 as its FRET pair

    (IFuA546/A647) (Supplementary Fig. S1). IFuA546/A647 showed a pH sen-

    sitivity from pH 5 to 6.5, suitable for studying pH variations in theEE/SEs, LEs and TGN, with typical intraorganelle pH values asindicated by the green arrowheads in Fig. 1e. ITfA488/A647 showed atransition from pH 5.5 to pH 7, spanning the EE/SE and RE pHvalues, with typical intraorganelle pH values indicated by thepurple arrowheads in Fig. 1f.

    Each DNA nanomachine maps pH of its targeted pathwayWhen the scFvfurin chimera was expressed inside HeLa cells, twoclosely spaced bands near the expected size (4345 kDa)(Supplementary Fig. S2) indicated that the fusion protein mimicsthe maturation of endogenous furin (Supplementary Fig. S3ac)17,and it is also localized, like endogenous furin, to the TGN(Supplementary Fig. S3d)18. When scFvfurin-expressing HeLaand TRVb-1 cells were incubated with 500 nM IFuA488/A647 inthe external medium and imaged, we observed that IFuA488/A647 wasefficiently endocytosed into perinuclear compartments; thisdid not occur in untransfected or mock transfected cells

    Salt

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    Figure 1 | Programming of DNA nanodevices. a, Schematic of the DNA nanomachines that map the furin (Fu) and transferrin (Tf) pathways: IFu (top) and

    ITf (bottom). IFu consists of a nicked duplex and cytosine-rich overhangs and undergoes a pH-dependent conformational change due to i-motif formation

    (green segment), leading to high FRET between Alexa 546 (magenta circle) and Alexa 647 (blue circle) labels on the scaffold. A dsDNA domain on IFu

    (gre

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