NOVEMBER 1, 1993
`
`VOLUME 90
`
`NUMBER 21
`
`N051: Li
`Lil I'.1IA:.,L~;‘
`I
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`EF-'u.-+"-r?1- o
`
`'H
`
`E F THE
`
`ffoceedings
`National Academy
`of Sciences
`
`OF THE UNITED STATES OF AMERICA
`
`
`
`Page 1 of 8
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`ILMN EXHIBIT 1036
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`Proceedings
`0!‘ THE
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`National Academy
`of Sciences
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`OF THE UNITED STATES OF AMERICA
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`Qficer:
`qf the
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`Proceedings
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`Jau-Ia: B. WYNGA&IDiN. Fania! Sccnswry
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`

`

`Proceedings
`
`OF THE
`
`National _Academy
`of Sciences
`
`OF THE UNITED STATES OF AMERICA
`
`November 1, 1993
`
`Volume 90, Number 21
`
`pp. 9741-40410
`
`Table of Contents
`
`CONDENSED INFORMATION FOR CONTRIBUTORS
`
`AUTHOR INDEX
`I.
`
`Commentary
`
`Nitric oxide: Foe or friend to the injured brain?
`Dennis W. Cltoi
`
`974 1-9743
`
`Papers from a Colloquium
`
`Images of science: Science of images
`A. V. Crew:
`
`Overview of imaging science
`Robert N. Beck
`
`Chargecl—coupled detector sky surveys
`Donald P. Schneider
`
`Pattern recognition at the Fennilah collide: and
`Superconducting Supercollider
`Henry J. Frisch
`
`Model observers for assessment of image quality
`Harrison H. Barrett, lie Yao. Jannick P. Rolland.
`and Kyle J. Myers
`
`9744—9745
`
`9746-9750
`
`9751-9753
`
`9754—9'i‘5'l
`
`9'.'r'58 -9765
`
`Image processing: Some challenging problems
`T. S. Huang and K. Aizawa
`
`Color. contrast sensitivity. and the cone mosaic
`David Williams. Nobutoshi Sekiguehi. and
`David Brainartl
`
`Color appearance: The effects of illumination and
`spatial pattern
`Brian A. Wandell
`
`Nonlinear processes in visual pattern discrimination
`Hugh R. Wilson
`
`The intelligent camera: Images of computer vision
`W. Eric L. Grimson
`
`Shape and motion from image streams: A
`factorization method
`Carlo Tomasi and Talteo Kanade
`
`Issues of imaging science for future consideration
`Robert N. Beck
`
`9766-9769
`
`9770-97 '1'?
`
`9778—9?84
`
`9785-9790
`
`9795-9302
`
`9803 -9807
`
`
`
`Page 3 of 8
`
`

`

`Proc. Natl. Acad. Sci. USA
`Vol. 90, pp. 10700-10704. November 1993
`Chemistry
`
`Generation and screening of an oligonucleotide-encoded synthetic
`peptide library
`(encoded synthetic libraries/combinatorial chemistry /peptide diversity}
`
`MICHAEL C. NEEDELS, DAVID G. Jones, EMILY H. Tare, Gneooav L. HEINKEL, LYNN M. Kocneasrenoea,
`WILLIAM J. Dowea, RONALD W. B.-mnerr. AND MARK A. GAr_Lo1=*
`Afiytnax Research Institute, 4001 Miranda Avenue. Palo Alto. CA 94304
`
`Communicated by Peter G. Schultz. August 2. J99}
`
`We have prepared a library of = 10‘ different
`ABSTRACT
`peptide sequences on small, spherical (10-pm diameter) beads
`by the combinatorial chemical coupling of both I.- and D-amino
`acid building blocks. To each head is covalently attached many
`copies of a single peptide sequence and, additionally, copies of
`a unique single-stranded oligonuclaotide that codes for that
`peptide sequence. The oligonucleotide tags are synthesized
`through a parallel combinatorial procedure that effectively
`records the proc by which the encoded peptide sequence is
`assembled. The collection of beads was screened for binding to
`a iluorescently labeled anti-peptide antibody using a fluores-
`cence-activated cell sorting instrument. Those beads to which
`the antibody bound tightly were isolated by fluorescence-
`activatcd sorting, and the oligonncleolide identifiers attached
`to individual sorted beads were amplified by the PCR. Se-
`quences ol' the amplified DNAs were determined to reveal the
`identity of peptide sequences that bound to the antibody with
`high atlinily. By combining the capacity for information stor-
`age in an oiigonucleotlde code with the tremendous level of
`amplification possible through the PCR, we have devised a
`means for specifying the identity of each member of a vast
`library of molecules synthesized from both natural and unnat-
`ural chemical building blocks. In addition, we have shown that
`the use of flow cytometry instrumentation permits facile iso-
`lation of individual beads that bear Itigh-affinity Iigds for
`biological receptors.
`
`Ligands for macromolecular receptors can be identified by
`screening diverse collections of peptides produced through
`either molecular biological or synthetic chemical techniques.
`Recombinant peptide libraries have been generated by in-
`serting dcgcncrate oligonucleotides into genes encoding cap-
`sid proteins of filamentous bacteriophage (1-3) or the DNA-
`binding protein Lac I (4). These random libraries may contain
`>109 different peptides, each fused to a larger protein se-
`quence that
`is physically linked to the genetic material
`encoding it. Chemical approaches to generating peptide li-
`braries arc not limited to combinatorial syntheses usingjust
`the 20 genetically coded amino acids. By expanding the
`building block set to include unnatural amino acids.
`the
`accessible sequence diversity is dramatically increased. In
`several of the strategies described for creating synthetic
`peptide libraries, the different peptides are tethered to a solid
`support in a spatially segregated manner (5, 6). Large librar-
`ies of soluble peptides have been prepared as peptide pools
`using the “tea-bag" method of multiple synthesis. Active
`peptides within these degenerate mixtures are identified
`through an iterative process of screening and sub-iibrary
`resynthcsis (7. 8).
`
`Using the split-synthesis protocol pioneered by Furka er al.
`(9), Lam er al. (10) have prepared libraries containing ===10‘
`peptides attached to 100- to 200-um-diameter resin beads.
`The head library is screened by incubation with a labeled
`receptor: beads binding to the receptor are identified by
`visual inspection and are selected with the aid of a micro-
`manipulator. Each bead contains 50-200 pmoi of a single
`peptide sequence that may be determined directly either by
`Edman degradation or mass spectrometry analysis. In prin-
`ciple. one could create libraries of greater diversity using this
`approach by reducing the bead dimensions. The sensitivity of
`peptide-sequencing techniques is limited to ==l pmol, how-
`ever, clearly limiting the scope of direct peptide-sequencing
`analysis. Moreover. neither analytical method provides for
`straightforward and unambiguous sequence analysis when
`the library building block set is expanded to include 1)- or
`other nonnatural amino acids.
`We recognized that the products of a combinatorial peptide
`synthesis on resin beads could be explicitly specified if it
`were possible to attach an identifier tag to the beads coinci-
`dent with each amino acid-coupling step in the synthesis.
`Each tag would then convey which amino acid monomer was
`coupled in a particular step of the synthesis. and the overall
`sequence of a peptide on any head could be deduced by
`reading the set of tags on that head. We now describe the use
`of single-stranded oligonucleotides to encode a combinatorial
`synthesis on 10-pm-diameter polystyrene beads. Peptides
`and nucleotides are assembled in parallel, alternating syn-
`theses so that each bead bears many copies of both a single
`peptide sequence and a unique oligonucleotide identifier tag.
`We have generated an encoded synthetic library of some 8.2
`X 105 heptapeptides and screened it for binding to an anti-
`dynorphin B monoclonal antibody (mAb) D3139 (4). using a
`fluorescence-activated cell sorting (FACS) instrument to
`select individual beads that strongly bind the antibody.
`
`MATERIALS AND METHODS
`
`Reagents and General Methods. The rnonodisperse 10-mo-
`diameter bead material used in this work was a custom-
`synthesized macroporous styrene/divinylbenzene copoly-
`mer functionalized with a 1,12-dianrinododecane linker. All
`protected amino acids were obtained from Bachem.
`Parallel Synthesis of a 69-Base Oligonucleotide and the
`Peptide H-Arg-Gin-Plle-Lys-Val-Val-Tllr-NH; (RQFKVVT).
`The C-terminal seven amino acid fragment of the opioid
`peptide dynor-phin B was synthesized in parallel with a
`69-mer oligodcoxyribonuclcotidc (51708) on 10-,u.m-diameter
`beads. The sequence of ST08 was 5’-AIC BAA 1‘_C_T (2112
`[LAC ATC TCTATA CTA TCA TCA CC [TA TC CT AT TT
`
`The publication costs of this article were defrayed in part by page charge
`payment. This article must therefore be hereby marked “advertr'.remem"
`in accordance with 18 U.S.C. §1‘!34 solely to indicate this fact.
`
`Abbreviations: Fmoc. 9-fluorenylmethoxycarbonyl; DMT. di-
`rnethoxytrilyl; mAb. monoclonal antibody: FACS, fluorescence-
`activated cell sorting.
`‘To whom reprint requests should be addressed.
`
`10700
`
`
`
`Page 4 of 8
`
`

`

`Chemistry: Needels er al.
`
`Proc. Natl. Acad. Sci. USA 90 (1993)
`
`10701
`
`TT AC] CTC ACI QAC TIC CAI IQC AC-3’. Underlined
`portions of this sequence correspond to PCR-priming sites,
`while the region in italics is homologous to the primer used
`for sequencing this template. The 14-base sequence enclosed
`in brackets represents the coding region of the template.
`The heads were first treated with a mixture of succinitnidyl
`4-O-DMT-oxybutyrate (where DMT is dirnethoxytrity1;Mo-
`lecular Probes) and the Loxybenzotriazole ester of either
`N-Fmoc-2.4-dimethoxy~4'-(carboxyrnethyloxy)benzhydry-
`larnine (i.e.. the acid-cleavable Knorr carboxamide linker,
`where Fmoc is 9-fluorenylrnethoxycarbonyl) or N—Frnoc-
`Thr(:err-butyl)-OH (for noncleavable experiments). The ratio
`of Frnoc-protected atnino groups to DMT-protected hydroxyl
`residues on the beads was determined spectrophotometri-
`cally to be ==20:1. The beads were subjected to 20 cycles of
`oligonucleotide synthesis on an automated synthesizer using
`3'-0—methyl-N,N-diisopropyi phosphoramidites of the fol-
`lowing nucleosides: N5-benzoyl-5'-0-DMT-(7-deaza)-25
`deoxyadenosine (Berry and Associates, Ann Arbor, MI),
`N‘-benzoyi-5’-0-DMT-2’-deoxycytidine, and 5'-O—DMT-
`thymidine (Glen Research. Sterling, VA). The beads were
`then removed from the instrument and treated for 5 min with
`10% (vol/vol) piperidine in dimethylformamide to remove the
`Fmo-C protecting group. After coupling the first amino acid
`residue [N-Fmoc-Thrtrerr-butyl)-OH],
`the beads were
`treated with a tetrahydrofuran solution of acetic anhydride
`and 1-methylimidazole to cap any unreacted amines. All
`peptide coupling reactions were l'l.ll'I for 20 min and contained
`0.11 M Fmoc-amino acid. 0.1 M 0-(benzotriazol-1-yl}
`1.1.3,3-tetramethyluronium hexafluorophosphate. 0.1 M
`1-hydroxybenzotriazole. and 0.3 M diisopropylethylamine in
`dimethylformamide,/CH;Cl;. 1:1. The beads were then sub-
`jected to two cycles of nucleotide addition on the synthesizer
`(detritylation with trichloroacetic acid; tetrazole-catalyzed
`phosphitylation; capping with acetic anhydride: oxidation
`with iodine in acetonitrile/water). Sequential steps of amino
`acid coupling and dinucleotide addition were repeated until
`synthesis of the peptide sequence RQFKVVT and construc-
`tion of the oligonucleotide coding region had been completed.
`After an additional 35 cycles of oligonucleotide synthesis, the
`beads were treated sequentially with piperidine/dimethylfon
`marnide, 1:9 for 3 min; thiophenol/triethylamine/dioxane.
`1:2:2. for 4 hr; ethylenediamine/ethanol, 1:1, for 5 hr at 55°C;
`and trifluoroacetic acid/water, 20:1. for 1 hr to fully depra-
`tect both the peptide and oligonucleotide chains. In experi-
`ments using the acid-cleavable linker, the supernatant from
`the trifiuoroacetic acid de-protection reaction was concen-
`trated in vacuo, and the isolated crude peptide was then
`analyzed by HPLC using a Rainin reverse-phase C15 column.
`Construction ohm Encoded Library. The parallel synthesis
`chemistry outlined above was used in the construction of the
`library. The sites of peptide synthesis were differentiated
`from DNA-synthesis sites in this experiment by coupling to
`all beads a mixture of N-Fmoc-Thr(ter!-butyl)-oxybenzotri
`azole and succinimidyl 4-0-DMT-oxybutyrate, as has been
`described. Sequences of oligonucleotide tags in the library
`deviated from ST08 only within the coding region. The
`3’-conserved region of the oligonucleotide ST08 was first
`synthesized on a total bead mass of 35 ntg (#1335 X 103
`beads). The Fmoc protecting group was removed. and the
`bead mass was divided into seven equal parts. To each
`aliquot was coupled one of seven different a-N-Fmoc-
`protected amino acids [side-chain protecting groups are
`shown in parenthesis): Arg(NG-2,2,5,7,8-pentamethylchro-
`man-6-sulfonyl), Gln(trityl), Phe, Lys(rerr-butoxycarbonyl),
`Val, D-Va]. and Thrtrerr-butyl). Each part was then subjected
`to two rounds of automated oligonucleotide synthesis. The
`respective sequences of the appended dinucleotides that
`specified uniquely each different amino acid residue were
`TA. TC. CT , AT. TT. CA, and AC. The beads were then
`
`pooled and mixed thoroughly; the entire bead mass was then
`subjected to Fmoc deprotection. This cycle of head parti-
`tioning. peptide coupling. oligonucleotide-dirner synthesis,
`bead recombination, and Fmoc removal was repeated for a
`total of seven times. The final Fmoc protecting group was not
`removed. Rather, the pooled bead mass was subjected to 35
`cycles of oligonucieotide synthesis. The library was then fully
`deprotected as described above.
`FACS Analysis of Antibody Binding to Beads. A portion of
`the library (typically 0.5-2 mg of beads) was suspended in
`blocking buffer [phosphate-buffered saline (PBS)/1% bovine
`serum albumin/0.05% Tween-20] and incubated at room
`temperature for 1 hr. The beads were pelleted by centrifu-
`gation and resuspended in a solution of mAb D3239 (10
`,ug/ml in blocking buffer) (4). The suspension was incubated
`on ice for 30 min. pelleted by centrifugation. and washed with
`blocking buffer. Beads were then suspended in a solution of
`phycoerythrin-conjugated goat anti-mouse antibody (Molec-
`ular Probes) for 20 min on ice. The beads were washed in
`blocking buffer and diluted in PBS for delivery into the FACS
`instrument (Becton Dickinson FACStar"'"‘). Beads that had
`bound mAb D32.39 were identified by their acquired fluo-
`rescence.
`Individual beads from both the most brightly
`stained 0.17% of the library and from the region having the
`lowest fluorescence (#98%) were sorted into PCR microcen-
`trifuge vials.
`PCR of Bead-Bound Template and Sequencing of PCR
`Product. PCRs consisting of 45 amplification cycles were
`done with Taq polymerase (Perltin—Elmer) according to the
`manufacturer's instructions. The reactions contained dUTP
`and uracil DNA glycosidase (GIBCO/BRL) to prevent car-
`ryover contamination with soluble product from previous
`amplifications (11). Biotinylated PCR product from individ-
`ual reactions was isolated with streptavidin-coated magnetic
`beads (Dynal. Great Neck. NY). After alkaline elution of the
`nonbiotinylated strand and washing, each bead sample was
`treated with sequencing mixture. Dideoxynucleotide se-
`quencing was done by using the primer 5’-ATC TCT ATA
`CTA TCA-3’ (SPl5) and B5: polymerase (Bio-Rad). accord-
`ing to the manufacturer's instructions. except that a 1:100
`ratio of deoxy- to dideoxynucleotide triphosphates (Pharma-
`cia) was used.
`Determination of Peptide-Binding Affinities. Binding affin-
`ities of various peptides for mAb D3239 were measured in a
`competition binding experiment. A tracer peptide (LR-
`RASLGGGRRQFKVVT; 50 pM) containing the known epi-
`tope for mAb D3239 fused to a consensus substrate sequence
`for CAMP-dependent protein kinase was radiolabeled to high
`specific activity with [y-33P]ATP (12) and mixed with various
`concentrations of the peptide of interest (10 ,u.M to 1 pM). The
`peptide mixtures were added to polystyrene wells coated
`with mAb D3239 (0.1 pg/ml). Samples were incubated 2 hr
`at 4°C. the wells were washed with PBS. and the radioactivity
`associated with each well was counted and used to generate
`a competitive binding curve. Under the conditions of the
`assay the lC_=.o should be close to the dissociation constant
`(Kd) for the peptide.
`
`RESULTS
`
`Establishing a practical bead-based oligonucleotide-encoded
`peptide library methodology demands that several key tech-
`nical criteria be met. These criteria include (i) the develop-
`ment of mutually compatible chemistries for parallel assem-
`bly of peptides and oligonucleotides. (ii) the selection of bead
`material with appropriate physical characteristics, (iii) the
`facile isolation of small beads bearing ligands that bind a
`target receptor. and (iv) successful PCR amplification and
`sequencing of template DNA from single beads. We have
`found that the properties of 10-p.n1-diameter beads fashioned
`
`Page 5 of 8
`
`
`
`Page 5 of 8
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`

`

`10702
`
`Chemistry: Needels er al.
`
`Proc. Natl. Acad. Sci. USA 90 f 1993}
`
`olic ethylenediamine (debenzoylation of protected cytidine
`and 7-deaza-adenine residues). These mild. anhydrous ami-
`nolysis conditions did not adversely affect protected peptide
`sequences (19), which were deblocked using triflnoroacetic
`acid under standard conditions.
`The carboxyl-terminal region of opioid peptide dynorphin
`B (YGGFLRRQFKVVT) has been previously shown to
`represent the epitope of anti-dynorphin B mAb D3239 (4):
`the soluble heptapeptide RQFKVVT binds mAb D3239 with
`high affinity (Kg = 0.5 nM). To test the efficacy of our
`chemical methods. a parallel synthesis of this peptide and a
`69-base oligodeoxyribonucleotide was perfonned on orthog-
`onally differentiated beads bearing an acid-cleavable Fmoc-
`protected carboxamide (Knorr) linker. The beads were ex-
`posed to full oligonucleotide and then peptide-deprotection
`conditions. and the trifluoroacetic acid supernatant contain-
`ing the cleaved peptide was analyzed by reverse-phase
`HPLC. Fig. 1a shows that the crude peptide from the parallel
`synthesis consists of a single major component (coelutirlg
`with authentic RQFKVVT; data not shown) and that this
`crude product is not significantly different from that gener-
`ated in a control peptide synthesis in which no oligonucleo-
`tide chemistry occurred (Fig. lb}. Fig. 2 demonstrates that
`the integrity of the DNA template containing cl dA was also
`maintained through the course of the parallel synthesis chem-
`istry.
`Construction of a Large Encoded Combinatorial Library.
`An encoded library designed to contain 823,543 (77) different
`heptapeptides attached to 10-um beads was constructed by a
`combinatorial synthesis using the seven amino acids arginine,
`glutarnine, phenylalanine, lysine, vaiine, D-valine, and thre-
`onine. a-N-Fmoc-protected threonine and 0-DMT-protected
`y~oxybutyrate residues were first coupled to all the beads to
`provide the orthogonally differentiated amino and hydroxyl
`groups for this synthesis. Starting with a total bead mass of
`35 mg (1.75 X 103 heads) ensured that each peptide sequence
`appeared on e200 different beads in the library. Peptide
`microsequencing analysis of an aliquot of the library con-
`firmed that the seven amino acids were stochastically dis-
`tributed among every position of the degenerate heptapeptide
`mixture (note that L-valine and D-valine are indistinguished in
`the Edman degradation procedure).
`The binding of mAb D3239 to control beads and to the
`bead library was analyzed by flow cytometry. Fig. 3a shows
`that beads carrying the positive control sequence RQFKVVT
`-l
`
`3
`
`30
`
`b
`
`30
`
`'
`
`l
`
`min.
`
`§
`
`0
`
`E 4
`
`0
`
`FIG. 1. ReveI'sed—phase HPLC chromalograms of crude peptide
`RQFKVVT. Asterisks mark peak corresponding to authentic mate-
`rial. (a) Peptide synthesized in parallel with 69~mer oligonueleotide
`ST03. (b) Peptide from control synthesis (no nucleotide chemistry).
`
`from a macroporous styrenefdivinylbenzene copolymer and
`derivatized with a dodecylamine linker are generally satis-
`factory for this work. The amino group loading of these beads
`was estimated to be #100 pmol/g by exhaustive acylation
`with Fmoc-glycine. followed by piperidine cleavage of the
`Fmoc group and spectrophotometric quantitation of the
`released piperidine—dibenzofulvene adduct (em 2 7800
`liter-mol‘1-cm"). With 5 x 109 heads per g. this corresponds
`to a maximum peptide loading of “=20 fmol per bead. Acy-
`lation of the beads with a mixture of an appropriately
`protected amino acid and an to-hydroxy acid provided or-
`thogonally differentiated amino and hydroxyl groups from
`which the peptide and nucleotide chains, respectively. could
`be extended. The average stoichiometry of peptide to olig0-
`nucleotide per bead is controlled by varying the ratio of
`amino and hydroxy acids coupled to the initial bead mass.
`Test peptide syntheses (5-mers to 12-mers) on these beads
`equipped with a trifluoroacetic acid-cleavable Knorr linker
`(13) using standard Fmoc chemistry were found to proceed
`with high fidelity that was indistinguishable from syntheses
`perfonned on conventional peptide synthesis resin. as deter-
`mined by HPLC analysis of the crude cleaved peptide
`carboxamides (data not shown).
`Parallel Synthesis of Peptides and Oligonucleotldes. Parallel
`synthesis strategies require 0‘) the use of a set of protecting
`groups on the amino acids and nucleotide building blocks that
`are mutually orthogonal and (ii) that each of the polymer
`chains be stable to the reagents used in the synthesis and
`deprotection of the second chain. Although. in principle, a
`variety of protection/deprotection schemes could be envis-
`aged, we preferred to use Fmoc/ten-butyl protection on the
`peptide building blocks because of the extensive commercial
`availability of natural and unnatural amino acids protected in
`this manner. However. the ten-butyl-based peptide side-
`chain protecting groups require treatment with strong acid
`(typically trifluoroacetic acid) for removal, conditions that
`lead to rapid depurination of oligonucleotides containing
`either 2'-deoxyadenosine (dA) or 2'-deoxyguanosine (dG)
`(14). This problem has been circumvented by using 7-dea2:a-
`2’-deoxyadenosine (c7dA) for dA in the template oligonucie-
`otide tag. The glycosidic bonds of deazapurine nucleosides
`are resistant to acid-catalyzed hydrolysis (15), and oligonu-
`cleotides incorporating these monomers are faithfully copied
`by thermostable polymerases used in the PCR (16. 17).
`Although not used in this work, acid-resistant guanosine
`analogs could also be incorporated into the template DNA.
`5’-O-dimethoxytrityl 2’-deoxynucleoside 3’-(O—methyl-
`N.N-diisopropyDphosphoramidites were used in all parallel
`syntheses. The reagent (I2/collidine/H30/acetonitrile) used
`to convert the nucleotide phosphite intermediates to phos-
`pholriesters in the DNA-synthesis protocol was not found to
`adversely afiect either the readily oxidized residues try]:-
`tophan and methionine or any of the other protected amino
`acids used in this work (data not shown). Complete removal
`of the 5'—O-DMT group from the growing oligonucleotide
`chain was achieved in ===40 sec using 1% trichloroacetic acid
`in dichloromethane, whereas all of the acid-labile side-chain
`protecting groups used conventionally in Fmoc/ten‘-butyl
`chemistry were inert to treatment with 1% trichloroacetic
`acid for 1 hr. Quantitative deprotection of the cr-amino
`residues required 5- to 10-min treatment with piperidine/
`dimethylformarnide (10% vo1/vol) and also resulted in partial
`demethylation of the protected polynucleotide phosphotri-
`esters (fry: == 45 min). Control experiments indicated that any
`aberrant phosphitylation of the resulting phosphodiester spe-
`cies during subsequent nucleotide chain elongation was re-
`versed by the final oligonucleotide deprotection steps, as
`noted by other workers (18). At the completion of the parallel
`synthesis, the DNA was fully deprotected by treatment with
`thiophenolate (phosphate 0-demethylation) and then ethan-
`
`Page 6 of 8
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`

`
`
`Chemistry: Needels er al.
`
`Proc. Natl. Acad. Sci. USA 90 (1993)
`
`10703
`
`
`
`Particle Scatter
`
`Particle Scatter
`
`Particle Scatter
`
`3 2
`
`UI
`
`logfluorescence
`
`FIG. 3. Flow cytometric analysis of binding of mAb D3239 to
`10-p-.rn beads beating peptide and oligonueleotide. Approximately 105
`events are recorded in each experiment. Fluorescence intensity is
`shown on vertical axis. (a) A 1:1 mixture of underivatized flower
`population) and RQFKVVT (upper population) beads as negative
`and positive controls.
`(13) Binding of mAb to library. (c) Specific
`binding to library blocked by preincubation of mAb with 10 p.M
`RQFKVVT.
`
`improve the
`and the stringency of wash conditions will
`capacity to isolate only the 1'tighest~afl'mity ligands.
`
`DISCUSSION
`
`We have developed chemistry to prepare a highly diverse
`oligonucleotide—encoded synthetic peptide library on micro-
`scopic beads by combinatorial synthesis. While this work
`was in progress, the concept of an oligonucleotide-encoded
`chemical synthesis was proposed independently by Brenner
`and Lerner (20). More recently, two other groups have shown
`that an L-amino acid peptide strand may be used to encode
`the combinatorial assembly of molecular structures that are
`not amenable to direct sequence analysis (21, 22). It seems
`likely that constraints on the sensitivity and throughput ofthe
`Edman procedure will ultimately restrict the scope of this
`peptide-coding approach to analyzing libraries of litnited
`diversity.
`Encoding a combinatorial synthetic procedure with oliga-
`nucleotides provides a mechanism for addressing the major
`limitations of ambiguity and sensitivity encountered in the
`direct structural analysis of minute quantities of ligands
`isolated from large libraries. The high capacity of DNA for
`information storage can be exploited to archive the precise
`details of a library's construction. In the example above, we
`used a "codon" structure of two contiguous nucleotides
`comprising three bases (cl dA, dC, and T), capable of
`encoding a synthesis incorporating up to 32 = 9 amino acid
`building blocks (only seven were used in this library). Ifc7 dG
`were also included in the coding template, then a combina-
`torial synthesis using 1000 different monomers could be
`accommodated by using at ‘‘codon‘‘ size of just 5 nt (45 =
`1024).
`in using an
`A second outstanding advantage inherent
`oligonucleotide-based coding scheme is the ability to achieve
`
`in ACT
`
`3123455
`
`-70 bp
`
`—|nn-lg-I-I-C-I-lb-O-
`
`FIG. 2. Amplification and sequence analysis of oligonucleotide
`STOB synthesized in parallel with peptide RQFKVVT. ta) Ethidium
`bromide-stained agatose gel electrophoresis of products from PCR
`amplifications of 51118 template attached to single sorted beads.
`Lanes: 1 and 6. DNA markers; 2. c"'dA-containing template from
`parallel synthesis; 3, dA-containing template after I-hr treatment
`with 95% trifluoroacetic acid}5% H10; 4. untreated dA-containing
`template; 5, zero bead control.
`(iv) Sequencing gel of the PCR
`amplification product from an individual bead; DNA sequence of the
`template coding region is shown at right.
`
`and a 69-rner oligontlcleotide tag are strongly stained by the
`antibody, whereas blank beads are unstained. By contrast,
`only a small fraction of the encoded library bound with
`D3239 (see Fig. 3b). Analysis of 105 events indicated that
`===2% of the library stained above background levels. Signif-
`icantly, this binding to mAb D3239 was specific for the
`combining site, as it could be completely blocked by prelu-
`cubating the mAb with soluble RQFKVVT peptide (Fig. 3c).
`Individual beads from the library having fluorescence inten-
`sities comparable with the positive control beads were sorted
`into microccntrifuge tubes for tag amplification by PCR
`(beads with fluorescence in the top 0.17% of the population
`were collected). Nucleotide sequences were obtained from 12
`sorted beads, and the deduced peptide sequences are given in
`Table 1. Representative peptide sequences obtained from
`single beads having fluorescence that was not significantly
`above background are also tabulated for comparison.
`These data are consistent with an earlier study showing
`that the preferred recognition sequence of mAb D3239 is
`localized to the six-amino acid fragment RQFKVV of dynor-
`phin B (4). Interestingly, D-valine appears best tolerated at
`positions outside the consensus motif. The range of affinities
`of peptides that were selected (Kg fi= 0.3—1400 nlvl) was not
`unexpected, given the design of the binding assay—i.e.,
`bivalent primary antibody with labeled second antibody
`detection. We anticipate that manipulation of the binding
`valency (for example. directly labeled monovalent receptor)
`Table I.
`
`Amino acid sequences of peptides on beads that bind mAb D3139
`High fluorescence intensity
`Low fluorescence intensity
`Kg. nM
`Sequence
`Sequence
`Kg, mlvi
`0.29
`QQFKVVQ ( T )
`QTVTVKK [ T)
`>1
`4.3
`KQFKVTQI T )
`QQVQRQT ( T )
`>D.4
`8.8
`TQF‘l('v'TK[T)
`KTQvvQF'l '1‘)
`ND
`16
`TFRVFRVI T}
`QVTQVRV { T)
`ND
`76
`FRRQFRVI T )
`F‘lJln'T'JR‘ll'('l')
`ND
`340
`RQFKQVQ ( T J
`
`Sequence
`TFRQFKV l T }
`TTRRFRV l T )
`TVRQFI-('1‘['l')
`QVRQFKT [ T)
`RQFRTVQ I T)
`KQFKVTK ( T)
`
`K4. nM
`370
`410
`5&0
`1400
`ND
`ND
`
`(positive control)
`0.51
`RQFKVVT
`A library of peptide-beating beads was screened for antibody ligands by using an indirect fluores-
`cence assay and FACS instrumentation. Sequences from the highly fluorescent beads are aligned to
`show consensus with the D3239 epitope. Afiinities of selected soluble peptides for mAb D3239 were
`determined by RIA. ND. not determined‘. (Tl. threonine linker residue.
`
`Page 7 of 8
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`10704
`
`Chemistry: Needels er al.
`
`Proc. Natl. Acad. Sci. USA 90 (I993)
`
`tremendous levels of target amplification through the PCR.
`We are therefore able to work with tiny quantities of DNA
`template and, hence, to use solid supports of microscopic
`dimensions in our syntheses. This will facilitate the construc-
`tion and screening of libraries that far exceed the diversity
`accessible through other tethered synthetic library tech-
`niques. Moreover, these libraries will employ manageable
`quantities of bead material that can t