ATOGRAPHIC SCIENCE SERIES
`
`VOLUME 27
`
`AFFINITY
`CHROMATOGRAPHY
`
`Template Chromatography of Nucleic Acids
`
`and Proteins
`
`Unbound RNA
`
`1
`
`A
`
`‘
`
`P
`
`
`
`Radioactuvuty,cpm
`
`Fraction
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`Exhibit 21 O4P.age 1 , _,.
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`
`, __ . , Enzo Exhibyt 2194
`BD v. Enzo
`
`HerbertCSeéPfi37
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`Enzo Exhibit 2104
`BD v. Enzo
`Case IPR2017-00181
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`Exhibit 2104 Page 1
`
`

`

`Aff
`
`O
`
`Chrotnatography
`TE CHROMATOGRAPHY
`IC ACIDS AND PROTEINS
`
`Herbert Schott
`Universitiit Tubingen
`stitut fur Organische Chemie
`gen, Federal Republic of Germany
`
`MARCEL DEKKER, INC.
`
`New York and Basel
`
`Exhibit 2104 Page 2
`
`

`

`Library of Congress Cataloging
`
`in Publication Data
`
`[date]
`Schott, Herbert,
`Affinity chromatography.
`
`science
`
`v. 27)
`
`( Chromatographic
`Bibliography:
`p,
`Includes
`index.
`1. Affinity chromatography.
`2. Nucleic acids-(cid:173)
`I. Title.
`Separation.
`3. Proteins--Separation.
`[DNLM: 1. Chromatography,
`II. Series,
`Affinity.
`3. Proteins-
`& purification.
`2. Nucleic Acids- -isolation
`isolation & purification.
`Wl CH943 v. 27 / QU 25 S375a]
`QP519.9.A35S34
`1984
`574.19'245
`84-17461
`ISBN 0-8247-7111-7
`
`-
`
`COPYRIGHT© 1984 by MARCEL DEKKER, INC. ALL RIGHTS RESERVED
`
`or transmitted
`this book nor any part may be reproduced
`Neither
`in any form or by any means, electronic
`or mechanical,
`including
`photocopying,
`microfilming,
`and recording,
`or by any information
`storag·e and retrieval
`system, without permission
`in writing
`from
`the publisher.
`
`MARCEL DEKKER, INC.
`270 Madison Avenue, New York, New York
`
`10016
`
`Current
`10
`9
`
`printing
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`PRINTED
`
`IN THE UNITED STATES OF AMERICA
`
`Exhibit 2104 Page 3
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`

`

`Contents
`
`Preface
`
`Abbreviations
`
`INTRODUCTION
`
`2
`
`IMMOBILIZATION OF NUCLEOSIDE PHOSPHATES,
`NUCLEOSIDES, AND NUCLEOBASES
`
`I.
`II.
`
`Immobilization of Nucleoside Phosphates
`Immobilization
`of Nucleosides
`and Nucleobases
`
`3
`
`IMMOBILIZATION OF OLIGONUCLEOTIDES
`
`I.
`II.
`
`Terminals
`Covalent Binding of the Oligonucleotide
`Secondary Valence Binding of Polymer-Bound
`Oligonucleotides
`
`4
`
`IMMOBILIZATION OF POLYNUCLEOTIDES AND NUCLEIC
`ACIDS
`
`I.
`
`II.
`III.
`
`and Mechanical Forces
`
`via Physical
`
`Immobilization
`or Irradiation
`Immobilization Using Chemical Activation
`Special Methods
`for the Immobilization of RNA
`and Polyribonucleotides
`
`iii
`
`ix
`
`3
`
`3
`12
`
`15
`
`15
`
`19
`
`23
`
`2 3
`28
`
`41
`
`V
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`Exhibit 2104 Page 4
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`

`

`Special Methods
`
`41
`
`IMMOBILIZATION OF RNA
`111. SPECIAL METHODS FOR THE
`AND POLYRIBONUCLEOT
`IDES
`
`yeast
`of immobilization of high-molecular-weight
`The efficiency
`RNA on aminated
`silica carriers
`has been studied
`[ 449]. More
`than 90% of the RNA added was bound
`to the carrier
`in undegrad(cid:173)
`ed form. The procedure
`allows us to link as much as 2. 0 mg of
`RNA per gram of carrier.
`During nucleic acid hybridization
`( 65°C, 2 x SSC) in the presence
`of trace amounts of pronase,
`chipping
`off of RNA was observed
`only during
`the first 6 hr of
`incubation.
`More than half of the immobilized poly(A) RNA re(cid:173)
`mained
`tig·htly bound
`to the carrier.
`The immobilized messenger
`and ribosomal yeast RN As were hybridized
`with total yeast
`[ 1251]
`DNA. Several methods
`for the immobilization
`of polyribonucleo(cid:173)
`tides
`take advantage
`of the vicinal diol groups present
`or poten(cid:173)
`tially present
`in polyribonucleotides.
`
`A.
`
`Immobilization of Oxidized Polyribonucleotides
`
`can
`groups of polyribonucleotides
`terminal 3'-hydroxyl
`The free
`in Sec.
`be oxidized
`in aqueous
`solution by periodate
`as reported
`I .B of Chap. 2 for the oxidation of mononucleotides.
`The result(cid:173)
`ing aldehydropolyribonucleotides
`form Schiff bases with the pri(cid:173)
`mary amino groups of AE-cellulose
`( Scheme 16). The unstable
`Schiff bases can be converted
`to stable
`structures
`by reduction
`The binding
`using NaBH 4 [8,103-105],
`in these products
`is
`ring
`thought
`to arise from the formation of a substituted morpholine
`structure
`that
`includes
`the nitrogen
`of the AE-cellulose
`and the
`five atoms
`that orig·inally constituted
`the ribose
`ring of the ter(cid:173)
`minal nucleoside.
`The assignment
`of this
`linkage
`is based on the
`of the produ-ct obtained
`from the borohydride
`reduction
`structure
`of the complex
`formed between periodate-oxidized
`AMP and meth(cid:173)
`ylamide
`[ 106] .
`to a hy(cid:173)
`may also be added
`The aldehyde-polyribonucleotides
`support
`to form a stable hydra(cid:173)
`drazine or a hydrazine-containing
`zone bond
`( Scheme 16). For example,
`a mixture of agar and poly(cid:173)
`acrylic
`acid hydrazide
`has been used
`in this way to immobilize
`tRNA [107].
`Periodate-oxidized
`mRNA has been coupled
`to a lin(cid:173)
`ear polymer of acrylic hydrazide
`entrapped
`in agar
`[ 108] . The
`reduction with NaBH 4 has also been used
`to stabilize
`the linkage
`in a similar complex obtained
`from periodate-oxidized
`tRNA and
`hydrazinyl-Sepharose
`[109].
`of RNA by its 31 terminus
`The attachment
`accomplished
`[ 110] using alkaline
`activation
`
`to agarose has been
`of agarose with CNB r,
`
`Exhibit 2104 Page 5
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`

`

`42
`
`Immobilization of Poly nucleotides
`
`/Nucleic Acids
`
`R~~-ovoJ
`H
`i + NaI04
`
`+~
`
`NH2 NH
`
`2
`
`QL
`+~tri
`
`0
`ct-NH-NH
`--N --NH
`2
`2
`
`RNAp
`
`~~~~:j
`
`""t{lfj
`
`I
`I
`Hl'-4 l~H
`~t' ma r1 x
`
`+ NaBH4t
`
`~
`matrix
`
`B ; nucleo base
`
`Scheme 16
`
`acid methyl ester of the aga(cid:173)
`followed by coupling of aminocarpoic
`rose. The caproyl ester agarose was then converted
`to the corres(cid:173)
`ponding hydrazide,
`After blocking
`resin carboxyl groups with
`glycinamide
`and water-soluble
`carbodiimide,
`the treated
`agarose
`is coupled with RNA that had previously
`been oxidized with sod(cid:173)
`ium periodate.
`Although
`rather
`complicated,
`this procedure may
`have specialized
`use.
`
`Exhibit 2104 Page 6
`
`

`

`Special Methods
`
`43
`
`porous
`such as silochrome,
`On the basis of inorganic materials
`glass,
`and chromosorb,
`a number of supports
`have been synthe(cid:173)
`sized which have aminopropyl
`and hydrazide
`groups.
`As a result
`of attachment
`of periodate-oxidized
`coli tRNA to these
`Escherichia
`materials,
`preparations
`were obtained having as many as 100- 900
`Az60 units per gram of sorbent.
`To avoid nonspecific
`sorption
`that might arise due to excess aminopropyl
`groups,
`the prepara(cid:173)
`tions of immobilized nucleic acids were treated with acetic anhy(cid:173)
`dride,
`The supports with immobilized
`tRNA thus obtained were
`shown
`to be almost completely
`inert with respect
`to heterological
`DNA [ 111].
`immobilized on
`tRNA and 5S, 16S, and 23S rRNA were readily
`agarose-dihydrazide
`[112,113],
`the amount coupled decreasing
`with increasing molecular
`size. Epichlorhydrine-activated
`Sepha(cid:173)
`rose gels were reacted with adipic acid dihydrazide.
`Oxidized
`RNA or nucleotides
`are coupled
`to hydrazide
`residues
`linked by
`a spacer
`to the derivatized
`Sepharose
`[ 114] .
`
`B. Reversible Complexation of Ribonucleotides
`Dihydroxyboryl
`Residues
`
`with Immobilized
`
`are capable of
`in ribopolynucleotides
`Terminal cis-diol groups
`containing
`coval(cid:173)
`forming
`specific cyclic complexes with supports
`ently bound dihydroxyboryl
`groups
`(Scheme 17). N-[N'-(m-di(cid:173)
`hydroxyborylphenyl)
`succinamyl] AE-cellulose
`prepared
`from the
`condensation
`of N- (m-dihydroxyborylphenyl)
`succinamic
`acid with
`AE-cellulose
`forms cyclic boronate
`structures
`at pH 8- 9 with mole(cid:173)
`cules containing
`the ribonucleoside
`diol group
`[ 115-118] . At pH
`
`matrix
`
`~q+
`
`Hif OH
`
`B ; nucleo base
`
`matrix
`
`pH >8
`
`pH <7 ~-0,
`)~
`
`R
`
`R ~ CH20H £!: oligonucleotide
`
`Qf ribonucleic
`
`acid
`
`Scheme 17
`
`Exhibit 2104 Page 7
`
`

`

`44
`
`Immobilization of Poly nucleotides
`
`/Nucleic Acids
`
`immobi(cid:173)
`of the
`the recovery
`6, the complex breaks down, allowing
`lized ribonucleotides
`from the support, which is then available
`for the binding of another
`ribonucleotides.
`Similar complex form(cid:173)
`ation can be observed
`on dihydroxylboryl-substituted
`methacrylic
`acid-polymer
`(borate
`gel)
`[ 119].
`It should be pointed out that
`from its chemical constitution,
`the borate gel appears
`to be more
`resistant
`than
`the borate-substituted
`cellulose
`to degradation
`by
`heat or by microorganisms.
`Moreover,
`these gels are suitable
`for
`chromatography
`under pressure,
`which is advantageous
`for pre(cid:173)
`parative
`separations.
`The uses of boronic acids
`immobilized
`to
`insoluble
`supports
`include
`separating
`ribonucleotides
`from deoxy(cid:173)
`ribonucleotides
`[119,120],
`isolating
`3'-terminal
`polynucleotide
`fragments
`from large RN As [ 121], and separating
`3'-modified
`polynucleotides
`from unmodified ones
`[ 122] .
`Immobilized boronic
`acid can also be used
`to separate
`aminoacyl-tRNA
`from unacylated
`species
`[116,119,123-125].
`the 31-terminal oligoribonu(cid:173)
`cellulose,
`Using dihydroxyboryl
`cleotides of the in vitro T3 RNA polymerase
`transcript
`has been iso(cid:173)
`lated
`[ 126] . The advantages
`and disadvantages
`of several bor(cid:173)
`onate-containing
`supports
`have been
`investigated
`[ 127] , with the
`following
`results.
`Boronate
`cellulose binds macromolecular RNA
`but often
`is not useful
`for trace amounts of material because of
`nonspecific
`adsorption.
`It was found
`that boronate polyacryla(cid:173)
`mide and boronate
`agarose both display very
`low levels of non(cid:173)
`specific adsorption.
`Boronate polyacrylamide
`specifically
`binds
`cis-diol-containing
`mononucleotides
`or short oligonucleotides,
`but
`does not retain polyribonucleotides
`sufficiently
`tenaciously
`for
`preparative
`use. Boronate
`agarose proved
`to be suitable
`for the
`fractionization
`of trace amounts of macromolecular RNA, but not
`for mononucleotides.
`
`Exhibit 2104 Page 8
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`