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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.
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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.
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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
.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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11
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
.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
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12
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
.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
https://doi.org/10.1101/2020.12.03.410936http://creativecommons.org/licenses/by-nc-nd/4.0/
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13
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
.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
https://doi.org/10.1101/2020.12.03.410936http://creativecommons.org/licenses/by-nc-nd/4.0/
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14
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.
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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
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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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