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`ILMN EXHIBIT 1043
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`Page 1 of 11
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`ILMN EXHIBIT 1043
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`

`
`Current Opinion in Chemical Biology
`Editor Benjamin G. Davis UK
`
`Volume 26 (2015)
`
`Editorial Enquiries
`
`Current Opinion in Chemical Biology
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`Current Opinion in Chemicai Bioiogy
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`Editorial Board
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`2015 Contents
`
`The subiect of chemical biology is divided into
`nine major sections, each oi which is reviewed
`once a year. Each issue contains one or two of the
`sections, and the amount of space devoted to
`each section is related to its importance.
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`February
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`Omics
`Edited by Benjamin F Cr-avatt and Thomas Kcciadek
`
`April
`
`Biocatalysis and biotransformation
`Edited by Thomas Ward and Valentin Kohler
`Bioinorganio chen-iistrtl_
`Edited by Nico Hi Lim and ‘(I LU
`
`June
`
`
`Next generation therapeutics
`Edited by Dario Neri and Jorg Scheueimann
`
`August
`
`I
`_
`I
`Molecular imaging
`Edited by Samie Jalirey and Atsushi Miyaweiki
`
`October
`
`_
`_
`Synthetic biology
`Edited by Michelle Chang and Huimin Zhao
`Synthetic biomolecuies I
`'
`Edited by Peng Chen and Christian H:ickenber9'3'
`
`December
`
`‘
`Energy
`Edited byTimo1hy Bugs and Mime‘ Rem“
`Mechanistic biology
`Edited by Paula Booth and Lynne Regan
`
`Duilio Arigoni iswilzcfland]
`Frances Arnold iusni
`Jacqueline K. Barton tusni
`Hagan Bayley iui<i
`Stephen J. Benkoyic [usm
`Carolyn Bertozzi {USA}
`Ronald Breslow iusni
`Thomas Cech rusni
`Jason Chin iuiq
`Benjamin F. Cravatt iusm
`William F. DeGrado (USA)
`Anne Dell ruio
`Peter B. Deryan (usiti
`Alan R. Fersht iLii<)
`Matthew Francis iusm
`Michael Gelb iusni
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`Item Hamachi Uaparli
`Cliuan He iusn)
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`Donald Hilvert (Switzerland)
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`Barbara lmperiali iusm
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`Chaitan Khosia iusiti
`Judith P Klinman iusiii
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`Stephen J. Lippard iusit}
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`Mohamed Mahariel {Germany}
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`Tetsuo Nagano uapan;
`Dario Neri (Switzerland)
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`Page 3 of 11
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`
`l-ELSEVIERI
`
`Available online at www.sciencedirect.com
`
`ScienceDirect
`
`Volume 26. June 2015
`
`CONTENTS
`
`Abslractedlindexed in: B|OSlS, CAB Abstracts international, CAB Health, Chemicai Abstracts, EMBASE, Medline.
`Also covered in the abstract and citation database Scopus". Full text available on ScEenoeDirect""
`
`Jérg Scheuerrinann and Dario Neri
`
`Editorial overview: Next generation therapeutics: Creating and
`exploiting the chemistry of large numbers
`
`Next generation therapeutics
`Edited by Dario Neri and Jorg Soheuermarin
`1
`
`Kyungmoo Yea, Jia Xie. Hongkai Zhang, Wei Zhang and
`Richard A Lerner
`Selection ol multiple agonist antibodies from intracellular
`combinatorial
`libraries reveals that cellular
`receptors are
`lunisticnally pleiotropic
`
`Claudio Zambaldo. Sofia Barluenga and Nicolas Winssiriger
`PNA-encoded chemical libraries
`
`John Mccaflerty and Darren Schofield
`ldentificalion of optimal protein binders through the use of
`large genetically encoded display libraries
`Gang Li. Wenlu Zheng. ‘ring Liu and Xiaoyu Li
`Novel encoding methods lor DNA-templated chemical libraries
`
`Tomomi Morioka. Nikita D Loil-:, Christopher J Hipolito,
`‘(uki Goto and Hiroaki Suga
`Selection-based discovery of maorocyclic peptides for the next
`generation therapeutics
`
`William H Connors, Stephen P Hale and Nichofas K Tenet-i
`DNA-encoded chemical libraries of macrocycles
`Moreno Wichert and Nikolaus Krall
`Targeting carbonic anhydrase IX with small organic ligands
`
`1
`_
`Alix I Chan. Lynn M McGregor and David R Liu‘
`Novel selection methods for DNA-encoded chemical libraries
`
`Peter Blakskjaer. Tara Heitner and Nils Jakob Vest ll-Ianaen
`Fidelity by design: Yocloreactor and binder "'=‘~F3 9”_"9h""'°”'
`ior small-molecule DNA-encoded libraries and drug discovery
`
`_
`_
`‘
`Florent Samain and Giulio Casi
`3.-nail iargeiad gylgtgxics from DNA-encoded chemical libraries
`Anthony D Keefe. Matthew A Clark. Ch|'i5*°l3hE" D “upp-
`Alexander Litoirchick and ‘ring Zhang _
`Chemical ligation methods for the tagging Ol DNA'€|1C°d'3d
`chemical libraries
`
`I
`_
`Christian Heinis and Greg Winter _
`Encoded libraries of chemically modified petptldefv
`
`I
`1
`I
`Jéirg Scheuermann and Dario Neri
`Dual-pharmacophore DNA-encoded chemical llbfflrlefi
`
`Mark J Wigglesworth. David C Murravi CEEUOIY" J B'a“"‘efi"
`Michael Kossenjans andJ Willem M Nissink
`_
`|.-.¢;;easing me cteliuery of next generation therapeutics from
`high throughput screening libraries
`
`The Cover
`An agoniat antibody induces neural cells from human bone marrow
`CD34-l» stem cells. This is an example of an antibody from an
`intracellular combinatorial
`library that
`induces lT_3n5'd'ff“’-”3”i'm‘°n'
`(Green: Tuji, Red: F—actin. Blue: Nucleus). FOF more '"l°"“a"°” 9'99 ‘he
`review by Kyungmoo Yea et al.. this issue-
`
`DOI 10.101M51367-5931(‘i5}00059-9
`
`
`
`Page 4 of 11
`
`

`
`
`
`Eisei/ten’
`
`Available online at www.sciencedirect.com
`
`Sciencebirect
`
`
`
`Novel selection methods for DNA-encoded chemical
`libraries
`Alix I Chan‘, Lynn M Mceregori-2 and David R Liu
`
`® CrossMark
`
`Driven by the need for new compounds to serve as biological
`probes and leads for therapeutic development and the growing
`accessibility of DNA technologies including high-throughput
`sequencing. many academic and industrial groups have begun
`to use DNA—encccled chemical libraries as a source of bioactive
`small molecules. In this review. we describe the technologies
`that have enabled the selection of compounds with desired
`activities from these libraries. These methods exploit the
`sensitivity of in vitro selection coupled with DNA amplification
`to overcome some of the limitations and costs associated with
`
`conventional screening methods. In addition, we highlight
`newer techniques with the potential to be applied to the high
`throughput evaluation of DNA~encoded chemical libraries.
`Address
`
`Department of Chemistry and Chemical Biology and Howard Hughes
`Medical Institute, Harvard University. 12 Oxford St, Cambridge. MA
`02133, United States
`
`Corresponding author: Liu, David R (drliu@fas.harvard.edu)
`‘These authors contributed equally to this work.
`9 Present address: Department of Cellular and Molecular Pharmacology.
`University of California. San Francisco. CA 94153, United States.
`
`current Opinion in Chemical Biology 2015, 26:55-61
`
`This review comes from a themed issue on Next generation
`therapeutics
`
`Edited by Dario Neri and Jéirg Scheuermenn
`
`For a complete overview see the I=ss._ue and the Egg
`Available online 24th February 2015
`http:l!cix.doi.orgil10.1016r‘i.cbpa.2015.02.D10
`
`136?’-5931.i"u 2015 Eisevier Ltd. All rights reserved.
`
`Introduction
`
`The itipidiycx|‘i:tntiii1;_5\\'c;i|ti1 oi‘i',t:mmiit'. |'Jl'(>[L‘t}|‘I1iL‘.:llt(i
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`in vitro selection methods for ligand discovery
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`atdtlirionnl Ftllllttiifii of .'1‘L‘iL‘L'Ii(i[1 or i’(}R '.1I1tpiiii<.".I1‘i(il1 oi‘
`their ii?-£§il)L‘i'tilftf(i DNA iIL'l1]}1i:l1'l.‘.‘o IlI‘lti
`i1!:l.‘~i3~.'i\'L:i)‘ parnllcl
`itigit-titriitlgliptlr i)Nr\
`.‘-'(.‘.l|LlCl](_'i|]_L{.
`i)(.‘t)(.'1'il.iil1;_r‘ on tilt:
`tot-.ii
`Iihrziry size. ptitattivu hits cam i1(.'
`iLi(.‘|1i.'ii'iL‘t.‘i
`fmni
`ziinpiificii sctiiiciiccs citilcr by L‘t):In|tail'i1‘l,'_{ tin: .‘\'L2(||.l(..'l1L‘L.'
`:iinmt|:lncc [ii that in the: st:irtin,<_{ iihr ‘y I17] or hy fitting
`(Ii).‘:t:I'\’L‘ti
`.‘iL‘(]lll‘..‘I1L'L‘ columns to :1 1‘lI.,‘,‘.{1IFi\'L‘ laintimiui Liih‘[l‘i—
`hution [8] or to thc: Poisson Lii‘i|.”1‘ii}lili(JI1 [18].
`
`iicici:tion:i on !x‘(liiLi—,*i1ip|‘J:ilTt.‘Li pmrcin
`While tr:ttiitii:m1|
`targets oi'iE:I‘ tiraimzttic-.iii'3-'
`iI'ICl'L‘:l.‘~tCti Cii:lL'iCItL'}‘ cn|np:lI'cii
`tn scrccttti. they inu~at hi: pcrforincii on 1| puriiicti t:ii';_r,ct
`protein oi‘ ilttcrcst. 'i‘;lr,i_'_ct
`il1'll'l'l(3i)iii‘/.ii[i{tll may rcemit in
`:lrtiii1L‘tt1zIi
`i)inLiitt_Lr_ or in Iiil: itififi of l1tl[i\'t.‘ ctn1i'1)I'n'l:ltion:ii
`}1I'(J|)(:l'[it.“il'i]:l[:i|'C|'C(]tlil‘Cl.i Fnrimittl i'i[iC iiimiit1p,tni1;iti\'c
`['.tl',‘J_l‘..'f\‘.
`in :lti(iilit.'m,
`\\':t3si]iIt}_'_ and Ciitlitlll stops rcqiiircti
`iiir scicctiorts tlsilig immoiiiiizcti iI:lI‘gt_'[h‘ Ina)‘ niso ti.‘m<i\'i:
`uctit-‘c iiiiratry mcinhchi cu‘ i':iii to rcmit in thc imizitinn oi‘
`ticsircti
`:\'Ii(_'L‘iL‘.*i.
`intonictioI1—(]cpt.:|1ti::I'it
`i‘('.R iii3i‘(IR'i
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`tn ;uit|rc.-;s
`titcsc iinlit-.ltitm.~i iiliti :::l‘.Ii)ic silmiit-.mco1I>c c\':lilI;itiun oi‘
`
`Current Opinion in Chemical Biolo; 2015. 5£55--671
`
`Page5of11
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`Page 5 of 11
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`

`
`56 Next generation therapeutics
`
`Figure 1
`
`(3)
`
`[39 Sequence
`’”\uv"“\w"m‘«.iir'""\.. )0
`
`binding compounds
`retained
`
` lp/m
`jar MM
`
`Dwpremoded library
`
`-
`-
`~.,_,~,.“". '”f;°::§e'§‘;'\T;:'5
`
`PCFI amplification and
`Idenllllcalion oi biriciing compounds
`
`(bi
`
`DNA-linked iiganarsi
`hairpin formation
` N/W ligand-larger bir:di'ng induces
`fl,\u/fl,\w,,,.W#,,,\_.
`/ ——'
`Wmplumomm mm“
`“W WW
`0 b C
`.=antibodyorself-labelingprotein domain
`
`extension at
`hairpin 3: and
`?*
`
`DNA-linked iargetisi in
`mammalian cell iysare
`
` m
`—* wmmwmmww
`F-‘CF-l amplification and DNA sequencing Io
`decode ligand-izinii.-i binding pairs
`
`i :~::iii-0
`
`MM
`_. mmmm
`.
`.
`.
`.
`.
`.
`FOR amp|'“cat'°n and
`identification oi binding compounds
`
`Exol
`Gages“-on 0,
`nonb-Niels
`_.
`
`hi‘
`Z.
`
`‘VV“WO
`photocrosslinking occurs
`upon small mmecuge binding
`
`_
`_
`irnmcaiiied
`protein target
`
`.
`(G) DNA-encoded iibraf)’
`wwwnotg
`wmiitioto ’
`f
`pc.DN,.4 * _ phommacnve
`moiety
`
`(d) DNA-dupisxlibraryol
`phermacophores
`
`fl ‘O
`
`immobilized
`ia.-get
`To-—
`
`Synlhesize
`
`m\w pharmacophore
`
`Biochemical
`
`"""9E" "“”'j’“9
`flNw V = known binder
`
`lgcltflfimitillicaftign grid
`E egA::1:’cr:::ho.'_:s‘"g
`
`identiiic£_Ilion_o! highest-
`affinity binderlsl
`
`(9)
`
` -Q
` ®_.
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`DN;Hlnkedmrge1
`.
`
`binding compounds more likely to be
`co-compartmentalized in micelles
`
`binding,
`gpggqpgpfi
`dissociation‘
`emutsitication .
`—~
`
`t.
`DNA l.
`'93 ‘on
`<:—+
`
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`
`wmflwmwm
`‘
`break rnicelies WW
`<3“-
`sequencing, and
`PCR amplification,
`indsntiiicalion ot binders
`Current Opinion in Chemical Biology
`
` .
`
`Technologies tor the in viiro selection of DNA-encoded chemical libraries. {a} In solid-phase selection, all library members are simultaneously
`evaluated, Library members that are capable binding of an immobilized target protein are physically separated from inactive library members. lb)
`Interaction determination using unpurllied proteins (IDUP) takes advantage of the selective formation of a DNA hairpin between binding species to
`encode both protein and ligand on the same DNA strand. (c) DNA-programmed affinity labeling lDPAL} makes use of an o|igonuc|e0tide—coLIpleG
`photoreactive group (PC—DNA) that covalently labels only proteins bound by a library member. These complexes protect the corresponding DNA
`tag from digestion and allow its subsequent sequencing and decoding. (cl) Encoded se|f—assernbling chemical tibraries lESAC} use DNA
`hybridization to display multiple pharmacophores for interaction with a target protein. Tightly binding pharmacophore combinations guide the
`synthesis of high—affinity ligands based on the pharmacophore fragments. {(2) Library selections using binder trap enrichment utilize micelles to co-
`cornpartmentalize DNA tags associated with binding species.
`
`lll)I't1f'lC!~i of
`t|f(.‘()l}‘Il)lI1(.'l.l
`l)i|1tlin,i_r_tx:t\:ir<:Cri all I'I'lCI'I’Il)Ct'fi
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`ll"i;_51irc lb). In l[)|’(Jl{, prim-:ii'i—s-iii-iill ll1lJlCCl|lt: ltiiidiiig
`l}l’ll'l]_{.‘i cni:0tlin,i_i, IJN.-‘5\
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`
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`The ll)l‘(JR :lppm:icli was L‘X['Cl‘l(lL!tl
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`:ic[ion t|ct<:i'iiiin-.itinii
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`
`Current Opinion in Chemical Biology 2015, 26:55-61
`
`I
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`www_scienc:edirect,corn
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`Page 6 of 11
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`
`Selection methods for DNA-encoded chemical libraries Chan. McGregor and Liu 5?
`
`l?-0"]. By operating in cell lys-ates, Il)llP preserves post-
`translational modifications and interactions with endoge-
`nous binding partners, enabling the study of t.lif'fiet1lt-
`to-purify targets anti increasing the potential biological
`relevance of detected interactions. During IDIIP, target
`pr(3t<:ins are associated with D\'A oligonucleotitle tags
`either noncovalently using a DNA-iinlted antibody or
`covalently using a SNAl"—t-atz. In a model library at library
`binding
`experiment
`using
`combined
`libraries
`of
`262 DNA—lin|ted small moiceules anti 256 cell
`lysates
`expressing SNAP—taggetl targets, IDUI’ enriched all five
`known interactions highly, despite having affinities vary-
`ing from 0.2 nlvl to 3.2 p.1\rl [2(l"l. This method provides
`an efficient approach For rapidly evaluating the binding of'
`ligand libraries in cases in which purified proteins are not
`availallle or differ significantly from their native cellular
`counterparts.
`
`l.i antl coworkers developed another methotl for assaying
`binding of small—mo|ecules libraries to unmodified, non-
`immobili7.etl proteins by DNA-programmed affinity label-
`ing (DPAL) 12]]. in DPAI. (Figure 1c), unmodified pro-
`teins are mixed with small l)NAo|igonnclcotides bearinga
`5"
`azcidoplienyl pliotocrosslinlting moiety (l‘C—DNA).
`\rVhen a DNA-taggetl small molecule binds the target,
`DNA l1ybridi:tation to a complementary region on the
`short PC-D.\'/\ brings the photo-reactive group into prox-
`imity with the protein target. UV irradiation leads to
`covalent attachment of the PC-DNA to the target protein.
`which can be identified by mass spectrometry. The DPAI.
`tcelinique was adapted to identify bintlers from libraries of
`small molecules by taking advantage of‘
`liybridi/.-ation
`between the PC-DNA anti the binding ligands‘ DNA tags
`[Z2"l. The DNA tags ofnon—binding small molecules are
`digested by I-Ixonueleasc I, but the hybridbced i)_\iA tags of
`bound moleculesare protected from digestion. in addition,
`DPAL iscompatible with targets in cell lysates, a condition
`in which targets may closely mimic their native state,-.1nd
`with large excesses of non-binding DNA—linlted small
`molecules. DPAI. can also perform iterative rounds of
`selection to greatly amplify DNA sequences corrcspontiirig
`to binding small molecules.
`
`Photo-aifinity probes, such as diazirines, bt:I1:/.0pl}entJnes, anti
`phenyl amides, stabilize interactions between protein targets
`and l)i\lr'\-lil1l(t‘.(_l small molecules [2l.23}. Using DPAL, Li
`and coworl-ters tzntielictl DNA setpienees etzrrespiitiding to
`interactions as wealt as 14 ml\l. lispeeially when combined
`with multivalent ligand display [23], the use ofphotoaffiniry
`probes could :~:t:Ibili7.e weal-: interactions enough to enable
`DNA-encotleci fiagiiierit-littsctl screens [24].
`
`ligand is hybridizctl to a library of DNA-encotletl pl1arma-
`cophores. The l1yl)l"lLll7.l:i.l mixture is suhjectetl to or t,'r'Im
`selection either to identify i'r-.1gments that improve bi ntling
`ofzl known ligand (affinity maturation} or to discover novel
`synergistically liinciing ligands. These fragnitints are then
`covalently coupled using a variety oi" |inl~'.ers. and the
`resulting compounds are assayed without the DNA har-
`eodes to discover linker arcliitectures optimal For binding.
`Using ESAC, Neri and eoworlters discovered l1igber—al"i'inity
`inhibitors of trypsin [26] anti l\-IX-Il’—3 [‘)'].
`
`When DNA-linltcti antibotlies are hound to ‘.1 protein or
`protein complex,
`the DNA tags are brought
`into close
`proximity anti can be llI]l\'C(l by ligation [27] or extension
`[28]
`to give a selectively amplifi-able DNA sequence.
`Landcgren and coworlters have validated the proximity
`ligation assay (PIA) for the t|1It1nl:ifit:t1tion of biomarlters
`by liigli-throtigliput sequencing [29] anti for the analysis
`of protein—protc-in interactions and protein snbcellnlar
`localizcation or post-translational modification [30,31].
`PLA was also applied to the study of small molecules
`capable of tlisrllpting the interaction between VE(Il’—:‘\
`and its receptors Vi€GFR—l
`and Vi-‘.(}i“R—Z [32].
`
`Using a microarray to quantify unique DNA tags for each
`antibody, PLA can evaluate all pairwise protein—protein
`interactions {Pl’is) within a larger set of proteins [33].
`Recently. Huang and coworkers applied l-’|.:'\ to inter-
`rogate I204 i’l‘ls in a one-by—one fashion [3-4-].rest1ltiiig
`in the identification ofhundrcds ofpreviousiy validated
`i’i’ls. Tagging individual antibodies with unique
`DNA barcodes could enable readout of Pl'ls by high-
`thronghput DNA setpiencingand expedite PPI catalog-
`ing efforts.
`
`Whereas the previously described methods use DNA
`liyliritlization to promote amplification of active library
`members, or
`tzifm selection typically results in spatial
`separation ofactive library members from inactive species.
`To select binding molecules from their DNA-encoded
`tzhemicnl
`library [I], Vipergen developed a technique
`called binder trap enrichment (Figure 1e) (infill/1’
`vipergerrcom). .=\i'ter exposing a l)NA—encotletl chemical
`library to a target of interest, binding pairs are compart-
`mentalizted in waterand oil emulsion droplets. Statistically.
`binding ligands are more efficiently co—conip-artmentalizcetl
`with the target protein than nonl'unctiona| library mem—
`bers.
`l*ln7.yInatic ligation of the t:o—compartmentalized
`olignnncleotitlesencoding the ligand and target is followed
`by PCR amplification and high-tlirotighput DNAsequcne—
`ingto-idt:t1til'y the binding entitle:-3.
`
`in an alternate approztcll to iragmenl:-l)ased selections, Neri
`and coworkers developed a DNA-liybtidization based
`approach for perfi)rming selections using encoded self—
`assembled clicntical lillraries (ICSAC) [25]. in 2111 ESAU
`selection (Figure id).
`at
`IJNA-linltetl small-molecule
`
`Reaction-discovery using DNA-encoded
`chemical libraries
`interest in the development of novel chemical transl’or—
`nrations has led to the development of unbiased reactivity
`screens l)-asctl on l.(I,r'l\lS 35l\ GCfl\'lS [30]. and santlwich
`
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`Page7of11
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`"Current Opinion in Chemical Biology 2015, 26:55—6i
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`Page 7 of 11
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`
`58 Next generation therapeutics
`
`immiinoassay [37]. Reaction discovery using DNA-
`encoded ehemi all
`libraries can dramatically streamline
`the reaction discovery process by enabling simiiltaneous
`evaluation of all possible eornbinzitions of potential reac-
`tants
`amon;_r_
`S1]l}‘i[l'ilEC library members. The First
`l_)l‘-."A-cnct)ded reaction discovery scliemes by Liu anti
`coworlters relied on solid—phase separation of bond—i'orin-
`ing pairs of library members and their encoding DNA
`from inactive species lliigure 2a) and resulted in the
`discovery of a l’d(ll)—metliated alltynamide-alltene cou-
`pling reaction [38], a Aiilllll-eat-aly:r.ed and acid-eaitalyzcd
`alltene hydroarylation [39] and a biocompatible, Ru(ll)-
`eataly?.etl azide reduction induced by Visible light l4(J'].
`
`In reactivity-tlepcndent PCJR (RDPCR) (Figure Zb), ct)-
`valent bond formation