design of smart antibody mimetics with photosensitive switches - … · 2020. 12. 3. · 1 design...

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Designofsmartantibodymimeticswithphotosensitiveswitches

LianHe1,*,PengTan1,YunHuang2,3,*,andYubinZhou1,3,*Dr.PengTan,Dr.LianHe,Prof.YubinZhouCenterforTranslationalCancerResearch,InstituteofBiosciencesandTechnology,TexasA&MUniversity,Houston,TX77030,USAEmail:[email protected];lhe@tamu.eduProf.YunHuangCenterforEpigeneticsandDiseasePrevention,InstituteofBiosciencesandTechnology,TexasA&MUniversity,Houston,TX77030,USAEmail:[email protected],Prof.YubinZhouDepartmentofTranslationalMedicalSciences,CollegeofMedicine,TexasA&MUniversity,Houston,TX77030Keywords:Optogenetics,nanobody,monobody,proteindesign,syntheticbiology,baseediting

ABSTRACT

Astwoprominentexamplesofintracellularsingle-domainantibodiesorantibodymimeticsderived

fromsyntheticprotein scaffolds,monobodiesandnanobodiesaregainingwideapplications in cell

biology,structuralbiology,syntheticimmunology,andtheranostics.Weintroducehereinagenerally-

applicable method to engineer light-controllable monobodies and nanobodies, designated as

moonbodyandsunbody,respectively.Theseengineeredantibody-likemodulardomainsenablerapid

and reversible antibody-antigen recognitionbyutilizing light. Byparalleled insertionof twoLOV2

modulesintoasinglesunbodyandtheuseofbivalentsunbodies,wesubstantiallyenhancetherange

of dynamic changes of photo-switchable sunbodies. Furthermore, we demonstrate the use of

moonbodies or sunbodies to precisely control protein degradation, gene transcription, and base

editingbyharnessingthepoweroflight.

.CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under apreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in

The copyright holder for thisthis version posted December 4, 2020. ; https://doi.org/10.1101/2020.12.03.410936doi: bioRxiv preprint





















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1.INTRODUCTION

Intracellularsingle-domainantibodies(intrabodies)andtheirmimeticsderivedfromsynthetic

proteinscaffolds,asmostnotablyexemplifiedbynanobodiesandmonobodies,aregainingwide

useincellbiology,structuralbiology,syntheticimmunology,andtheranostics.[1-6]Single-domain

intrabodiesandtheirmimeticssuchasmonobodiesornanobodiesrivalconventionalantibodies

by theirsubstantiallysmallersizes (12-15kDavs150-160kDa), freedomfromdisulfide-bond

formation,andeaseofinvitroandincellularexpression.Recentengineeringeffortshaveledto

the generation of several classes of chemically or light-dependent single-domain antibodies,

eitherbasedonsplitnanobodies (optobody)[7] orhybridproteins thatutilizeaphotosensitive

switch (OptoNB and OptoMB)[8-9] or circularly permuted bacterial dihydrofolate reductase

(LAMA).[10]Nontheless,theseengineeredintrabodiesexhibitarelativelyslowactivationkinetics

orsufferfromarelativelylowormodestdynamicrangeoflight-inducedchanges.

Bearing these unmet needs in mind, we introduce herein a set of smart monobodies and

nanobodiesthatrespondtolightwithinseconds,intheformofON-switches(sunbody)orOFF-

switches (moonbody). The light-oxygen-voltage domain 2 (LOV2) derived from Avena Sativa

phototropin1hasbeenengineeredintointrabodiestoconferallostericcontrolofproteinactivity

bylight.Wegreatlyimprovedtheperfomanceofourlight-controlledmoonbodiesbyoptimzing

theLOV2insertionintheEFloopratherthantheDEloopasdescribedintheOptoMBtool[9].By

optimizing LOV2 insertion sites and paralleled insertion of two LOV2 modules into a single

sunbody, we significantly enhanced the dynamic range of light-inducible response when

comparedtotheexistingOptoNB[8].Withthesesmartsunbodiesormoonbodies,wedemonstrate

theirwideapplications in theremotecontrolofprotein localization,celldeath, transcriptional

programmingandprecisebaseediting.



https://doi.org/10.1101/2020.12.03.410936http://creativecommons.org/licenses/by-nc-nd/4.0/

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2. RESULTS AND DISCUSSION

2.1DesignofMoonbodyasLight-ControllableAntibodyMimetics

We first setout todesigna light-controllablemonobody (termedmoonbody)by inserting the

LOV2photoswitchintoafibronectintypeIIIdomain(FN3)scaffoldthatspecificallyrecognizes

theSrcHomology2 (SH2)domainofAbelson tyrosinekinase (Abl).[11-12] Six insertion sites at

exposed loop regions (Supplementary Figure 1a) were selected, with three situated in the

antigen-recognizingBC,DE, FG loops (the equivalent of complementarity-determining regions

(CDRs) seen in an antibody) and the other three at the opposing loops (Figure 1a-c). We

envisionedthatlight-inducedconformationalchangesinLOV2wouldallostericallymodulatethe

moonbody-targetinteraction,therebypermittingphotoswitchablecontrolofthemoonbody.To

visualizethelight-dependentchangesinmoonbody-SH2associationinlivecells,weanchoredthe

SH2domaintothenuclearenvelope(NE)viafusionwithmEmerald-laminA,andco-expressed

theengineeredmoonbodyasacytosolicproteinwithpartialdistributioninthenucleoplasm(NP).

InsertionofLOV2atSite4(DEloop)abolishedthemoonbody-targetinteractionregardlessofthe

presenceof light(SupplementaryFigure1b-c), likelyowingtothedisruptionof theantigen-

bindingpocket.TheinsertionofLOV2atSite2(BCloop)ledtoanappreciableincrease(

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antigen upon light stimulation. We further validated the transferability of our engineering

approachbyusingtwoadditionalmonobodies thatrecognizethesmallubiquitin-likemodifier

protein (SUMO) and the maltose-binding protein (MBP).[13-14]. Both engineered moonbodies

showed light-induced dissociation from their corresponding target proteins (Supplementary

Figure 2). These moonbodies might find broad applications in the cost-effective elution or

enrichment of recombinant MBP- or SUMO-fusion proteins by switching on and off light,

respectively.

2.2.Spatiotemporalcontrolofantigenrecognitionandtargetdegradationwithmoonbody

Thegenerationofmoonbodyallowedustocontrolantibody-antigenrecognitionwithhighspatial

andtemporalprecision.Weconfirmedthefeasibilityofspatialcontrolbyalternativelyfocusing

the photostimulation upon two individual cells under the same imaging field (Figure 2a). As

anticipated,onlythecellwithintheilluminatedareashowedlight-dependentdispersionofNE-

boundmoonbodyintothenucleoplasm;whereastheothercellwithoutphotostimulationshowed

no appreciable changes in the subcellular distribution of moonbody (Figure 2a and

SupplementaryMovie1).Whenthewholeimagingfieldwasexposedtolightstimulation,both

cellsshowedsimultaneouslight-dependentmoonbodyredistribution(SupplementaryMovie1).

Temporally,themoonbody-targetinteractionturnedouttobereversible,asreflectedbyrepeated

NE-to-NP translocation of moonbody in response to at least 10 dark-light cycles of

photostimulation(Figure2bandSupplementaryMovie2).Theactivationanddeactivationhalf-

livesweredeterminedtobe7.8±0.1sand46.5±0.3s,respectively(SupplementaryFigure1d).

Together,modular insertionofLOV2 intoanFN3-derivedmoonbodyenablesnoninvasiveand

reversiblecontrolofsingle-domainantibodymimeticsbylight.

Wefurtheraskedwhethermoonbodycouldbeutilizedtoconditionallyfine-tunetheexpression

levels of its binding target. To test this,we fused the SH2-specificmoonbodywith the auxin

signalingF-box2protein(AFB2),acomponentintheSkp1-Cul-F-Box(SCF)E3ubiquitinligase




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complexthatcanrecruitauxin-inducibledegronsforproteasomaldegradation.[15]Moonbodycan

directlyrecognizeitstarget,thusobviatingtheneedforauxinandthefusionofthedegrontagto

atargetprotein.Wereasonedthatthelight-triggeredmoonbody-targetdissociationcanprevent

SH2frombeingdestroyedbytheproteasomaldegradationmachinery(Figure3a).Indeed,inthe

presenceofescalatingdosesofpulsedlightstimulation,weobservedagradualrecoveryofSH2-

mEmeraldsignalsinthetransfectedcells(Figure3b),thusestablishingthefeasibilityofusinga

photoswitchablemoonbodytomodulatethetargetproteinexpressionlevelsinlivecells.

2.3.DesignofSunbodyasPhotoactivatableNanobody

Wenext extended the similar engineering approach, aswell as theNE translocation assay, to

screenphoto-switchablenanobodies(designated"sunbodies“;Figure4a-b).WeusedamCherry-

specificnanobody(LaM8)[16]asatestcaseandinsertedLOV2intoflexibleloopregionsopposing

theCDRs(Figure4bandSupplementaryFigure3a),thelatterofwhichareinvolvedindirect

antigen recognition and thus remain unperturbed. The constructs S1, S2, and S4 exhibited

negligibleorlittlechanges(

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light-triggered translocation towardNE (t1/2, on = 2.6 ± 1.8 s; Figure5). The sunbody-antigen

associationwasalsofoundtobereversible(Figure5a).Notably,theuseofadimericconcatemer

ofsunbody(2xsunbody)couldfurtherenhancethestrengthoflight-switchableantibody-antigen

binding, as reflected by >2-fold increase in the signal-to-background ratio reported by four

additional subcellular translocation assays (from the cytosol to mitochondria [Mito], plasma

membrane [PM], endoplasmic reticulum [ER], or early endosome [EE]; Figure 5b-d and

SupplementaryMovies3-4).Thesefindingsclearlyestablishthefeasibilityofusingsunbodyto

interrogateproteinslocatedatdifferentsubcellularorganelles.

2.4.Sunbodyforphoto-controllablegenetranscriptionandbaseediting

We thenmoved on to explore the use of sunbody for remote control of gene expression.We

combinedsunbodywiththeFLAREplatform[17]tocreateaSolarFLAREsystemforlight-inducible

transcriptional activation (Figure6a). Inour envisioneddesign, light stimulation initiates the

translocation of a cytosolic anti-mCh sunbody-TEV hybrid protein toward PM-tetheredmCh-

FLARE components (mCh-LOV2-TCS-tetR-VP16), and brings TEV in close proximity to the

exposedsubstrate(TEVcleavagesiteorTCS)tocleavethepolypeptide,ultimatelyreleasingthe

otherwisePM-restrictedtranscriptionalcoactivator(tetR-VP16)intothenucleitorecognizethe

nucleotide tetracycline operator (tetO) sequence and activate gene expression. The

photosensitive LOV2modules embodied in both sunbody and the FLARE system confer tight

control over gene transcription by light. The photo-inducible gene transcription was first

validatedbyusingbluefluorescentprotein(TagBFP)asareporter(Figure6b).Inthedark,we

did not observe appreciable TagBFP signals, attesting to the strict control of the SolarFLARE

system.Bycontrast,amarkedincreaseofTagBFPsignalswasnotedinthelight-illuminatedgroup,

suggesting that the light-inducible antibody-antigen interaction effectively activates the

SolarFLAREsystemtodrivegeneexpression(Figure6b).WenextreplacedtheTagBFPreporter

with an N-terminal fragment of mixed lineage kinase domain-like pseudokinase (MLKL-NT;

residues1-182)thatiscapableofelicitingnecroptoticcelldeath(necroptosis),[18]withthegoalof




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developing an optogenetic suicide device. Upon light stimulation, we observed a substantial

increaseofcelldeath,asreflectedbytheappearanceofSYTOXbluestainingofthenucleiofdead

cellsafterPMpermeabilizationbyMLKL-NT(Figure6candSupplementaryFigure4).These

resultsestablishSolarFLAREasalight-controlledtranscriptionalprogrammingdevice.

Next, we set out to combine sunbody with the CRISPR/Cas9-mediated C-to-T base editing

technique[19]todesignaphotoactivatablecytosinebaseeditor(paCBE).Toachievethis,wesplit

thefunctionalunitsofCBEintotwoparts(Figure6d):PartIcontainsamCherry-taggedpartially

enzymaticallydisabledCas9(Cas9nickase,orCas9n)withsgRNA(mCh-Cas9n);whereasPartII

contains anti-mCh sunbody fused with the cytidine deaminase APOBEC1 and a uracil DNA

glycosylaseinhibitor(UGI)topreventU:GmismatchbeingrepairedbacktoaC:Gbasepair.[20]We

reasonedthat,uponlight-triggeredsunbody-mChinteraction,paCBEcouldrestoreitscytosine-

to-thymine editing function. To quickly test this idea, we used a “Gene ON” (GO) luciferase

reportersystem[21]tomonitortheactivityofpaCBEbeforeandafterlightstimulation(Figure6e).

TheGOsystembecomesactivatedonlywhenafunctionalCBEconvertsCtoTtocreateadenovo

ATGcodonat thebeginningofareportergene(e.g., luciferase), therebyenablingtranslational

initiationandsuccessfulproductionofthegene.Whenthetransfectedcellswereshieldedfrom

blue light, we did not observe notable bioluminescent signals. Upon photostimulation, we

detected a substantial increase of bioluminescence, presumably owing to the expression of

luciferaseafterC>Tconversionatthestartcodon(Figure6e).Collectively,ourresultsindicate

thesuccessfuldesignofaphotoswitchablebaseeditingsystembytakingadvantageofthelight-

induciblesunbody-antigeninteraction.

3.CONCLUSIONS

Insummary,byusingwidely-applicableintrabodiesasproteinscaffolds,wehaveillustratedthe

successfuldesignofaseriesofsyntheticsunbodiesandmoonbodies,intheformofON-switches




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(sunbody) or OFF-switches (moonbody), that respond to light within dozens of seconds.

Complementarytotherecentlydevelopedoptobodies,optoNBsandLAMAs,[7-8,10]ourengineering

effortsgreatlyexpandthesmartantibodytoolboxby(i)extendingtheoptogeneticengineering

approachtoantibodymimetics,and(ii)offeringswitchableintrabodieswithvaryingkineticand

dynamic features.Worthy tonote,wehaveenhanced the rangeofdynamic changesofphoto-

switchable sunbodies (over2-foldcompared tooptoNB)by taking twoapproaches:paralleled

insertionoftwoLOV2modulesintoasinglesunbodyandtheuseofsunbodyconcatemers.The

perfomanceofmoondies(optimizedLOV2insertionintheEF-loop)hasbeengreatlyimproved

comparedtotherecenltyreportedOptoMB.Giventhehighmodularityandfaciletransferability

of our engineering approaches, we believe that a growing number of smart antibodies and

antibodymimeticscanlikewisebegeneratedinthenearfuture.Thesemoleculartoolswillgreatly

facilitate the mechanistic dissection of cell signaling and accelerate the development of

personalized medicine, such as chimeric antigen receptor (CAR) T-cells and customized

therapeutic biologics that respond to drugs and beverage intake. In particular, beverage-

switchableantibodiesareofimmediatetranslationalvaluesgiventheirhighcompatibilitywithin

vivoapplicationsandtherelativelylowbarriertowardclinicaltrials.

4.EXPERIMENTALSECTION

Celllinesandcultureconditions:HeLaandHEK293T(humanembryonickidney)celllineswere

obtainedfromATCCandculturedunder37°Cata5%CO2atmosphere,andmaintainedinthe

Dulbecco'smodifiedEagle'smedia(DMEM,Sigma-Aldrich,StLouis,MO,USA),supplementedwith

10%fetalbovineserum(FBS).

Molecularcloningandplasmidconstruction:Thestandardrestrictionenzymedigestion-ligation

andNEBuilderHiFiDNAAssemblymethodswereusedtocreateplasmidsinthisstudy.TheKOD

StartDNApolymerase(EMDMillipore,MA,USA)wasusedforPCRamplification.Allthesubcloned




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sequenceswereverifiedusingdiagnosticrestrictiondigestionandSanger’ssequencinganalysis.

AlltheothercloningreagentswerepurchasedfromNewEnglandBiolabs(Ipswich,MA,USA).

cDNAsequencesencodingmonobodiesandnanobodiesusedinthisstudywerecodon-optimized

andsynthesizedasgBlockbyIntegratedDNATechnologies(IDTInc, IA,USA).Themonobody-

encodingcDNAs(SH2Abl,MBPandSUMO)wereindividuallyinsertedintoacustomizedpcDNA-

mCherryvectorbetweentheEcoRIandXbaIrestrictionsitestogeneratemCh-taggedmonobodies.

To create a nuclear envelope (NE)-targeting SH2abl, cDNA of lamin A was inserted into a

customizedmEmerald-C1vector,followedbySH2abl insertion(NheI-EcoRI).AsLOV2fragments

werePCRamplifiedandinsertedintomonobodiesbyusingtheNEBuilderHiFiDNAAssembly

method. Forphotoswitchabledegradation,moonbody (S5.1) cDNAwasamplifiedviaPCRand

then inserted into the pSH-EFIRES-P-AtAFB2-mCherry vector (Addgene, #129716) between

EcoRIandNotIsitestoreplacemCherry.

TomakeGFPfusednanobody,cDNAencodingtheanti-mCherrynanobodyLaM8wasclonedinto

the pTriEx-GFP vector between HindIII and XhoI sites to yield pTriEx-GFP-LaM8. AsLOV2

fragmentswere PCR amplified and inserted viaNEBuilderHiFiDNAAssembly. The construct

exhibitingthehighestlight-inducedchangeswasdesignatedas“sunbody”(S0+S3).Thetandem

sunbodyexpressionvector(2xsunbody)wasmadebyinsertingoneadditionalcopyofsunbody

intothepTriEx-GFP-sunbodyplasmid.Tomakemitochondria-targetingmCh,thecDNAsequences

encodinghumanAKAP11-30(flankedbyNheIandBamHI)wereinsertedintomCherry-N1toyield

AKAP1-mCh.ForERanchoredmCh,thehumanSac1fragment(residues521-587)wasclonedinto

thepEGFP-C3backbonebyutilizingtheEcoRI-KpnIrestrictionsites,followedbyGFPreplacement

bymCh(betweenNheIandXhoIsites).TheplasmamembranetargetingmChconstructwasmade

asAgeI-mCh-EcoRV-CAAX-XbaIinthesamebackbone.




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TocreateaSolarFLAREsystem for light-inducible transcriptionalactivation, theTEVprotease

component (from Ca-FLARE (protease), Addgene #92214) was assembled into pTriEx-GFP-

sunbody to yield GFP-sunbody-TEV. The mCh and LOV2-TCS (TEV cleavage site)-tetR-VP16

fragments (fromCa-FLARE (TF), Addgene, # 92213)moduleswere fused into a pcDNA3.1(+)

backbone tomakePM-anchoredmCh-LOV2-TCS-tetR-VP16. TheTagBFPorMLKL expression

cassetteusedintheSolarFLAREsystemwasmadebyputtingTagBFPcDNAorhumanMLKL-NT

(1-182)(EcoRI/XbaI)underthecontrolofatightTREpromoter.

For photoactivatable cytosine base editor (paCBE), mCh and Cas9n (Cas9-D10A nickase)

fragmentswereinsertedintoapcDNA3.1(+)vectorviaHiFiassemblytomakemCh-Cas9n(Part

I).FNLSHiFiwasreplacedbyGFP-sunbody in thepLenti-FNLSHiFi-P2A-Purovector(Addgene,

#136902) to make the APOBEC1-GFP-sunbody-UGI fusion construct (Part II). The luciferase-

based GO system with sgRNA in the same vector was obtained from Addgene (pLenti-mU6-

Luc2GO-PGK-Neo,Addgene#136905).

Celltransfection:DNAtransfectionwasperformedbyusingtheLipofectamine3000transfection

reagent (ThermoFisherScientific,MA,USA)according to themanufacturer’s instructions. For

live-cell fluorescence imaging experiments, cells were seeded in four-chamber 35-mm glass-

bottomdishes(D35C4-20-1.5-N,Cellvis,MountainView,CA,USA)onedaybeforetransfection,

and imaged 24-48 h after transfection in an imaging cage equipped with 5% CO2 with the

temperaturesetat37°C.

Live cell photostimulation, time-lapse imaging and imaging data analysis: Time-lapse confocal

imagingwasperformedonaNikonA1RconfocalmodulemountedontoaninvertedNikonEclipse

Ti-Ebody.Thelightsourcescamefromamulti-lineargonlasermodulecontaining405,488,561

and640nmlasers.Alive-cellimagingcagedplatformwasusedtomaintainthetemperatureat




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37oCwith5%CO2tokeepcellshealthyduringtheimagingprocess.A10x,20Xairobjectivelens

and40xor60xoilimmersionobjectivelenswereusedforimageacquisition.

Tomonitor light-induced association or dissociation between sunbodies/moonbodies and the

correspondingantigens,HeLacellsseededonglass-bottomdisheswereco-transfectedwiththe

indicatedplasmidsshownintherelatedfigures.Confocalimageswereacquired24-48hoursafter

transfection.Thecellswereimagedevery4secfor2nminunlessotherwisenoted.The488-nm

lasersourcetoexciteGFPwasalsousedforphotostimulation(with1-5%output).Toquantify

fluorescentsignalsatselectedareas,weusedtheregion-of-interest(ROI)toolboxinNikonNIS-

Elementssoftwaretodefinethenuclearenvelope(NE)ornucleoplasm(NP)regions.The“Time

Measurement”toolwasusedtodeterminethemCherryintensitiesformoonbodyvariantsand

GFP intensities for sunbody variants. The fluorescence intensity ratio (FNE/FNP) was used as

readout, with the changes in the ratio plotted as F/F0 or ∆F/F0. For spatially-restricted

photostimulation,theFRAPmoduleintheNikonimagingsystemwasused,withthe488-nmlaser

poweroutputsetat0.2%-5%.

Moonbodyregulatedproteindegradation:TheplasmidencodingthemoonbodyfusedwiththeF-

boxproteinatAFB2wasco-transfectedwithSH2-mEmeraldintoHeLacells.Cellsweretreated

withorwithoutacustomizedblue lightsource(470nm,40µW/mm2)after16htransfection.

Lightstimulationwasappliedfor10%(1minON,9minOFF),30%(3minON,7minOFF),or50%

(5minON,5minOFF).Afteranadditional16hours,cellswereimagedandSH2-mEmeraldmean

intensitywasmeasured.MoonbodywithoutatAFB2wasusedascontrol.

SolarFLARE system for gene expression:The construct encoding GFP-sunbody fusedwith TEV

protease (GFP-sunbody alone as a control) was co-transfected with PM-mCh-LOV2-TCS-tetR-

VP16andthepTRE-TagBFPreporterinHEK293Tcells.Eighthourspost-transfection,cellswere

exposedtopulsedbluelightstimulationfor16h(470nm,40µW/mm2)withalightcycleof1min




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ONand9minOFF.Cellskeptinthedarkwasusedasacontrol.Confocalimageswereacquired

after48hoursoftransfectionwitha10xor20xairobjectformCh,GFPandBFPchannels.Eight

fieldsofviewwererecordedforeachcondition.GFPexpressionareawasmaskedandthemean

TagBFP intensity was calculated in this mask, with areas outside GFP-positive areas used as

background.Thebackground-correctedmeanTagBFPintensitieswerecalculatedandplotted.For

light-induciblenecroptosis,thepTRE-TagBFPvectorwasreplacedbythepTRE-MLKL-NTplasmid,

with the remaining procedures and conditions identical to the SolarFLARE-BFP reporter

experimentdescribedabove.Tomonitorcelldeathinreal-time,livecellswerestainedwiththe

SYTOXbluedye(ThermoFisherScientific,S11348,1:5000dilution,Cf=1µM).

Luciferase reporter assay: To examine the efficiency of photoactivatable cytosine base editor

(paCBE), the mCh-Cas9n, APOBEC-sunbody-UGI, and pLenti-mU6-Luc2GO-PGK-Neo (as base

editingreporter)wereco-transfectedintoHEK293Tcells.Cellsweretreatedwithbluelight(470

nm,40µW/mm2,1minON-9minOFFcyclesfor16h)orkeptinthedark8hoursaftertransfection.

Sunbody alone to replace APOBEC-sunbody-UGI was used as control. 72 h post-transfection,

bioluminescencemeasurementswereperformedbyusingaBright-GloLuciferaseAssaySystem

fromPromega(catalog#:E2610)bydirectlyaddingreagentstotheculturemediumata1:1ratio.

Fiveminuteslater,theluminescencesignalswerequantifiedbyusingaCytation5CellImaging

Multi-ModeReader(BioTek,Winooski,VT,USA).

Statisticalanalysis:AllthedatawereplottedusingtheGraphPadPrism8.3.0graphingsoftware.

Quantitativedatawereshownasmean±s.e.m.unlessotherwisenoted.Theanalyzednumber(n)

ofsamplesweredescribedinthefigurelegendsforeachexperiment.Thehalf-livesandmedian

effectiveconcentrations(EC50)weredeterminedbyusingtheGraphPadPrismsoftwarepackage.

ACKNOWLEDGEMENTS.WethankthefinancialsupportsfromtheNationalInstitutesofHealth

(R01GM112003 to YZ, R21GM126532 to YZ, R01CA232017 to YZ, R01HL134780 to YH, and




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R01HL146852 to YH), the Welch Foundation (BE-1913-20190330 to YZ), the John S. Dunn

Foundation(toYZ),andtheAmericanCancerSociety(RSG-16-215-01-TBEtoYZandRSG-18-043-

01-LIBtoYH).

AUTHORCONTRIBUTIONS

YZandYHconceivedtheideasanddirectedthework.LH,PTandYZdesignedthestudy.LHand

PT performed the experiments. LH and PT analyzed the results. YZ, YH and LH wrote the

manuscript.Alloftheauthorscontributedtothediscussionandeditingofthemanuscript.

COMPETINGINTERESTS

Theauthorsdeclarenocompetinginterests.

FIGURES




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Figure1.Designandoptimizationoflight-switchablemonobody(moonbody).

a. Schematicdepictingthedesignoflight-switchablemonobody(designated“moonbody”)and

the nuclear envelope (NE) translocation assay used for screening. A photoswitch LOV2 is

insertedintoselectedloopregionstoenablephoto-inducibletargetrecognitioninareversible

manner. Light-dependent shuttling of moonbody between NE and the nucleoplasm

(quantifiedastheNE/NPratioofmChsignals)ismonitored.Yellowcirclesrepresentthethree

CDR(complementarity-determiningregion)-likeloopregionsthatmediatemoonbody-target

recognition.

b. LOV2 insertion sitesmapped to the3D structure of an anti-SH2Ablmonobody (PDBentry:

3T04).Even-numberedinsertionsiteswerecreatedinthetarget-recognitionloops,whereas

odd-numberedsiteswerelocatedoppositetotheantigen-recognizingBC/DE/FGloops.

c. 2Dtopologyrepresentationofananti-SH2monobody,withtheinsertionsitesindicatedby

circles.Themonobody-LOV2 junctionregions forS5or itsvariantswereshownbelowthe

Dar

kLi

ght

mCh-moonbody(Anti-SH2Abl)

NE-SH2Abl-Emerald (NE-Ag) Merge

a

d

b

S1

S4

S2

S3S5

S6BC loop

FG loop

DE loop

N

CLOV2 insertion sites

c

e

SH2Abl

N

C

ABFG C D

S1

S2

S3

S4 S6

DE loop

BC loop

FG loop

moonbody

E

S5

TATISGLSPGSGLATT...EAAKELGSGGVDYTITVTATISGLSPGSGLATT...EAAKEL---GVDYTITVTATISGLSPGSGLATT...EAA------GVDYTITV

LOV2S5S5.1S5.2

MonobodyEF loop

MonobodyEF loop

WT S1 S2 S3 S4 S5 S5.1 S5.2 S6

Ligh

t-ind

uced

cha

nges

in

the

NE/

NP

ratio

(mC

h; Δ

F/F 0

, %)

0

-10

-20

-30

-40

-50

10

Dark

Light

Antigen(SH2Abl-Emerald-lamin A)

Moonbody(Monobody + LOV2)

Nuclear envelope (NE)

Anti-SH2Abl moonbody LOV21-73 74-103

Nucleoplasm (NP)

15

cartoon.SeeSupplementaryFigure1fordetailedsequenceinformationofallconstructstested

inthestudy.

d. Quantificationoflight-dependentresponses(astheNE/NPratio)ofmoonbodyvariants.See

SupplementaryFigure1forrepresentativeimages.InsertionatSite5(S5)ledtothehighest

light-inducedchange.n=6-25cells.Dataareshownasmean±s.e.m..

e. Representativeconfocal imagesofaHeLacell co-expressingananti-SH2moonbody(mCh-

taggedvariantS5.1;red)andNE-tetheredSH2domainofAblkinase(NEtethered-antigenor

abbreviatedasNE-Ag;green)inthedarkorafterlightilluminationfor10sec.Scalebar,10

µm.

16

Figure2.Spatiotemporalcontrolofmoonbodybylight.

a. Spatial control of the moonbody-antigen interaction in live cells. HeLa cells were co-

transfected with NE-SH2Abl (as the Ag; not shown) and mCh-moonbody (shown in gray).

Photostimulation was sequentially applied to Cells 1 and 2 in the same imaging field as

indicatedbythebluebox.Scalebar,10µm.AlsoseeSupplementaryMovie1.

b. Temporal control of the moonbody-antigen binding in live cells. The nucleoplasmic mCh

intensity(asillustratedinpanelb)inresponseto10repeateddark-lightcyclesofstimulation

wasquantified.Photostimulationwasappliedbyusingthe488-nmlaserwith5%input.n=

11cells.AlsoseeSupplementaryMovie2.

Rel

ativ

e nu

cleo

plas

mic

mC

herry

inte

nsity

0 200 400 600 800 1000

Time (sec)

1.0

1.1

1.2 Light

ba Dark

S1

Focused illumination

S2

Step 1. Photostimulation on Cell #1

S1+S2

Step 2. Photostimulation on Cell #2

Step 3. Photostimulation on Cells #1 and #2

Spatial control

17

Figure3.Light-tunablecontrolofproteinturnoverbymoonbody.

a. Schematicillustratingtheuseofananti-SH2moonbodyforlight-tunabledegradationofthe

targetproteininmammaliancells.AFB2bindstheSkp1-Cul1-Rbx1toformaubiquitinligase

complex to mediate proteasomal degradation. Light-induced dissociation between the

moonbodyanditstargetcanbeexploitedtoconditionallycontrolproteindegradation.

b. Quantificationoflight-tunabledegradationofSH2-mEmeraldusingmoonbody.HEK293cells

weretransfectedwithAFB2-moonbody(ormoonbodyaloneascontrol)andSH2-mEmerald,

andtheneithershielded(Dark)orexposedto8-hblue light illuminationwith intensifying

pulses(withtheONandOFFdurationsindicatedinthex-axis).Anexternal470-nmLEDlight

wasusedas the light source (40µW/mm2). n=5 fieldsof viewper condition.Errorbars

denotes.e.m..

a b

Dark

Light

Proteosomal degradation

Cul1

Skp1AFB2

Rbx1E2

Ub

UbUbUbUb

Cul1

Skp1AFB2

Rbx1E2

Target(SH2-mEmerald)

AFB2-moonbody

SH2-mEmerald

SH2-

mEm

eral

d m

ean

inte

nsity

(a.u

.)

500

1000

1500

2000

moonbodyalone

AFB2-moonbody

Light pulse(ON:OFF)

0 1:9 0 1:9 3:7 5:5

18

Figure4.Engineeringphotoswitchablenanobody (sunbody) toenable light-controllable

antigenbinding.

a. Cartoon depiction of the design and the NP-to-NE translocation assay. Photoswitchable

redistribution of an engineered anti-mCherry (mCh) nanobody (designated “sunbody”) is

usedasthereadout.SunbodyisexpectedtoshuttlebetweenNEandNPinalight-dependent

manner.YellowcirclesrepresentthreeCDRsinvolvedinantigenbinding.

b. Insertion sites for LOV2 mapped to the modeled 3D structure of an anti-mCh nanobody

(LaM8).S1,S2andS4arelocatedattheoppositesideofCDRloops.BoththeN-terminus(S0)

and S3 are in closeproximity to CDRs. See Supplementary Figure3 for detailed sequence

information.

c. Quantification of light-induced changes in the NE/NP ratio for an anti-mCh GFP-tagged

sunbody.ThecombinationofLOV2fusiontotheN-terminus(S0)anditsadditionalinsertion

atS3ledtothestrongestlight-induciblechanges(S0+S3).SeeSupplementaryFigure3for

d

Dar

kLi

ght

GFP-sunbody(S0+S3)

mCh-lamin A (NE-Ag) Merge

c

Ligh

t-ind

uced

cha

nges

in

the

NE/

NP

ratio

(GFP

, ΔF/

F, %

)

CTRL S0 S1 S2 S3

S0 +

S3 S40

20

40

60

80

100

a b

Dark

Light

Nuclear envelope (NE)

Antigen(mCh-Lamin A)

Sunbody(Nanobody + LOV2)Nucleoplasm (NP)

1-18 19-126LOV2

1-46 47-126LOV2

LOV2 1-126

1-76 77-126LOV2

1-92 93-126LOV2

1-76 77-126LOV2LOV2

S0

S1

S2

S3

S4S0+S3

(Sunbody)S1

S3

S4

CDR1

CDR3CDR2

C

N

LOV2 insertion sites

S0

S2

19

light-inducedchangesofeachconstruct.n=15-66cellsfromthreeindependentassays.Data

areshownasmean±s.e.m..

d. RepresentativeconfocalimagesofaHeLacellco-expressingsunbody(GFP-taggedLaM8-S3;

green)andNE-tetheredmCh-laminA(red)beforeandafterlightilluminationfor10sec.Scale

bar,10µm.

20

Figure 5. Improved sunbody shows high sensitivity for light-dependent subcellular

targeting.

a. Quantificationof the sunbody-antigen interaction in response to three repeateddark-light

cycles.ThechangesinthenucleoplasmicGFPsignalswereusedasthereadout.n=23cells.

b-d. Sunbodyusedfor light-dependentsubcellulartargetingof itsbindingpartner.HeLacells

were transfected with an anti-mCh GFP-tagged sunbody (1x; green; top panels), or its

concatemericform(2x;green;bottompanels),alongwiththemChasantigen(red)tethered

toPM(b),ER(c),oroutermitochondrialmembrane(d).ThequantificationofrelativeGFP

signalsatthecorrespondingsubcellularorganellesbeforeandafterlightilluminationwere

shownnext to the images (n=15-75 cells). Theuseof 2xsunbody in a single construct

substantiallyenhancedthesignal-to-noiseratio.Scalebar,10µm.AlsoseeSupplementary

Movies3-4.

c

a

1x 2x

ER /

cyto

sol r

atio

(GFP

)

0

1

2

3

Anti-mChGFP-sunbody

mCh-Sac1 (ER-Ag)

1x

2x

DarkLight

Anti-mChGFP-sunbody

AKAP1-mCh(Mito-Ag)

1x

2x

1x 2x

Mito

/ C

ytos

ol ra

tio (G

FP)

0

1

2

3 DarkLight

1x 2x

PM /

cyto

sol r

atio

(GFP

)

0

1

2

3 DarkLight

Time (sec)0 20 40 240 260 280 480 500 520

1.0

1.5

2.0

2.5

t½= 2.6 ±1.8 s

Nor

mal

ized

cha

nges

of

NE

-GFP

sig

nals

Sunbody LOV2LOV2 1-76 77-1262

1x

2x

Anti-mChGFP-sunbody

mCh-CAAX(PM-Ag)

d

b

21

Figure6.Useofsunbodytoenablephotoactivatablegenetranscriptionandbaseediting.

a. CartoonillustratingthecombinationofsunbodywithamodifiedFLAREsystem(designated

SolarFLARE) toenable light-inducibleexpressionofgenesof interest, suchasTagBFPasa

reporterortheN-terminaldomainofMLKL(MLKL-NT)asanecroptosisinducer.

b. QuantificationofBFPexpressioninHeLacellstransfectedwithSolarFLARE(sunbogy-TEV+

FLARE)orthecontrol(sunbodyalone+FLARE)vectors,aswellastheTagBFPreportergene,

beforeandafterlightilluminationfor8h.n=10fieldsofviewfromthreeindependentassays.

c. QuantificationofnecroptoticcelldeathasindicatedbySYTOXbluenuclearstainingofdead

cells.HeLacellswere transfectedwithSolarFLARE(sunbogy-TEV+FLARE)or thecontrol

(sunbody alone + FLARE) vectors, as well as the inducibleMLKL-NT expression cassette,

a b c

Rel

ativ

e Ta

gBFP

inte

nsity

(Fol

d ch

ange

)

5

10

15

20 DarkLight

sunbodyalone

sunbody-TEV

0 Rel

ativ

e SY

TOX

blue

inte

nsity

(Fol

d ch

ange

)

5

10

15

20 DarkLight

sunbodyalone

sunbody-TEV

0Nuclear envelope

Dark

Light

SolarFLARE

TagBFP

MLKL-NT

Necroptosis (SYTOX+)

Sunbody-TEV

FLAREmCh

LOV2

TCS

VP16

Reporter

PM

d

2 4 6 100 8

sunbodyalone

paCBE

Relative luciferase activity(Fold change)

DarkLight

e

Light(base editing)

APOBECUGI

Luc

mCh-Cas9n

sgRNA

X

ACG

SFFV

Luc

ATG

SFFV

✓

APOBEC

UGI

C > T

APOBEC-sunbody-UGI

paCBE

22

beforeandafterlightilluminationfor8h.AlsoseeSupplementaryFigure4forrepresentative

images.n=10fieldsofviewfromthreeindependentassays.

d. Designofaphotoactivatablecytosinebaseeditor(paCBE).Uponphotostimulation,sunbody-

mChassociationre-assemblestwofunctionalunitsofCBE(PartI:themCh-Cas9n/sgRNAfor

genome targeting; Part II: APOBEC1-sunbody-UGI for C-to-T conversion) to restore the

activityofpaCBE.A“GeneON”(GO)luciferasereportersystemisusedtoreporttheactivityof

paCBEbefore and after light stimulation. Successful recruitment of Part II to the targeted

genomic locus is anticipated to cause C-to-T conversion in the start codon (ACG>ATG) to

initiatethetranslationofaluciferasereportergene.

e. QuantificationofthebaseeditingefficiencyofpaCBEbyusingluciferaseactivityasreadout.

Sunbodyalonewasusedasanegativecontrol.n=3independentassays.

Supplementarymaterialscontain:

SupplementaryFigs.1-4

SupplementaryMovies1-4

CaptionsforSupplementaryMovies

Supplementary Movie 1. Spatial control of the moonbody-antigen interaction in

mammalian cells. Time-lapse imaging of two HeLa cells co-expressing anti-SH2Abl mCherry-

moonbody(showningrey)andthenuclearenvelope-tetheredantigen(SH2Abl-mEmerald-lamin

A; not shown here). Sequential localized photostimulation (488 nm laser; 0.5% output) was

appliedasfollows:(1)Cell#1atthetop-rightcorner(0-12sOFF;16-49sON;59-129sOFF);(2)

Cell#2atthebottom-leftcorner(139-180sON;182-262sOFF);and(3)bothcells(271-290s

ON;300-390sOFF).

23

SupplementaryMovie 2. Temporal control of themoonbody-antigen interaction in live

cells.Time-lapseimagingoftwoHeLacellsco-expressingananti-SH2mCherry-moonbody(grey)

and the nuclear envelope-tethered antigen (SH2-mEmerald-lamin A; not shown). Pulsed

photostimulationat488nmwasappliedasindicatedbytheverticalbarsontherightgraph.The

fluorescentintensitiesoftwocircledregionsinbothcellswereplottedontheright.

SupplementaryMovie3.Time-lapseimagingofaHeLacellco-expressingtheantigen(Mito-

mCh;red,middle)andananti-mCherry,GFP-tagged2xsunbody(green,right).A488nm-

laserwith1%outputwasusedforphotostimulation.

Supplementary Movie 4. Light-induced protein translocation to different subcellular

organelles.Shownwas timelapse imaging of HeLa cells co-expressing an anti-mCherry, GFP-

tagged2xsunbody(green,middlepanels)andmCherryastheantigen(red,rightpanels),tethered

toER(toppanels),PM(middlepanels)orearlyendosome(bottompanels).A488nm-laserwith

5%outputwasusedforphotostimulation.

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