Protocols

Methods in Molecular BiologyJohn M. Walker, SERIES EDITOR

26. Protocols for Oligonucleotide Conjugates, edited b Sudhir Agrawal,

25. Computer Anal sis of Sequence Data: Part II, edited bAnnette M. Griffin and Hugh G. Griffin, 1994

24 . Computer Anal sis of Sequence Data: Part I, edited bAnnette M. Griffin and Hugh G. Griffin, 1994

23 . DNA Sequencing Protocols, edited b Hugh G. Griffinand Annette M. Griffin, 1993

22. Optical Spectroscop , Microscop , and Macroscopic Techniques,edited b Christopher Jones, Barbara Mullo ,and Adrian H. Thomas, 1994

21 . Protocols in Molecular Parasitolog , edited b John E. H de, 199320. Protocols for Oligonucleotides and Analogs, edited b

Sudhir Agra al, 199319 . Biomembrane Protocols : I. Isolation and Anal sis, edited b

John M. Graham and Joan A Higgins, 199318 . Transgenesis Techniques, edited b David M rph

and David A. Carter, 199317 . Spectroscopic Methods and Anal ses, edited b Christopher Jones,

Barbara M llo , and Adrian H. Thomas, 199316 . Enz

Elizabeth F. Ho nsell, 199313 . Protocols in Molec lar Ne robiolog , edited b Alan Longstaff

and Patricia Re est, 199212 . P lsed-Field Gel Electrophoresis, edited b MargitB rmeister

and Le Ulano sk , 199211 . Practical Protein Chromatograph , edited b Andre Kenne

and S san Fo ell, 199210 . Imm nochemical Protocols, edited b Margaret M. Manson, 19929 . Protocols in H man Molec lar Genetics, edited b

Christopher G. Mathe , 19918 . Practical Molec lar Virolog , edited b Mar K L. Collins, 19917 . Gene Transfer and Expression Protocols, edited b

Ed ard J. M rra , 19916. Plant Cell and Tiss e C lt re, edited b Jeffre W. Pollard

and John M. Walker, 19905 . Animal Cell C lt re, edited b Jeffre W. Pollard and John M. Walker, 19904 . Ne N cleic Acid Techniq es, edited b John M. Walker, 19883 . Ne Protein Techniq es, edited b John M. Walker, 19882. N cleic Acids, edited b John M. Walker, 19841 . Proteins, edited b John M. Walker, 1984

Methods in Molec lar Biolog ₁

Protocolsfor Oligon cleotide

Conj gatesS nthesis and Anal tical Techniq es

Edited b

S dhir Agra alWorcester Fo ndation for E perimental Biolog ,

Shre sb r , Massach setts

Fore ord b

H. Gobind Khorana

H mana Press Toto a, Ne Jerse

� 1994 H mana Press Inc .999 Ri er ie Dri e, S ite 208Toto a, Ne Jerse 07512

All rights reser ed.

No part of this book ma be reprod ced, stored in a retrie al s stem, or transmitted in an form or b anmeans, electronic, mechanical, photocop ing, microfilming, recording, or other ise itho t rittenpermission from the P blisher.

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Main entr nder title:Methods in molec lar biolog .

Protocols for oligon cleotide conj gates : s nthesis and anal tical techniq es / edited b S dhirAgra al,

p. cm.-(Methods in molec lar biolog ; 26)Incl des inde .ISBN 0-89603-252-31 . Oligon cleotides-S nthesis-Laborator man als.

2. Oligon cleotides-Anal sis-Laborator man als . I . Agra al, S dhir. II . Series : Methodsin molec lar biolog (Toto a, NJ) : 26.

[DNLM : 1 . Oligon cleotides-chemistr . W 1 ME9616J . 26]QP625.047P757 1993547.7'9-dc20DNLM/DLCfor Librar of Congress 93-23127

CIP

Preface

D ring the past ten ears or so, there has been an increasingn mber of p blications describing ario s aspects of oligon cleotidechemistr and the application of oligon cleotides in basic, as ell asapplied science . More recentl , ario s oligon cleotide conj gatesha e been s nthesi ed that are c rrentl being st died for theirpotential diagnostic and therape tic applications . With the introd c-tion of ne oligon cleotide conj gates, anal tical methods for theanal sis of these oligon cleotides are also being de eloped and opti-mi ed . The present ol me, Protocols for Oligon cleotide Conj -gates: S nthesis and Anal tical Techniq es, is designed to bringreaders p-to-date on the protocols for the s nthesis of ario s oligo-n cleotide conj gates and on all the important techniq es for theanal sis of s nthetic oligon cleotides and their analogs . The s n-thetic chemistr of man of these oligon cleotides and their ario sanalogs has been co ered in a companion ol me entitled Protocolsfor Oligon cleotides and Analogs: S nthesis and Properties.

The first chapter of Protocols for Oligon cleotide Conj gates de-scribes the protecting gro ps in oligon cleotide s nthesis. In thischapter, the a thor has described all protecting gro ps that ha e beensed to date in ario s stages of the de elopment of oligon cleotide

s nthesis, s pported b more than 400 references . The second chap-ter describes the s nthesis and incorporation of modified bases intooligon cleotides to introd ce reporter gro ps. Chapters 3 and 4describe the deri ati ation of s nthetic oligon cleotides ith aminogro ps and ith the thio gro ps, respecti el . In both chapters, pro-tocols ha e been pro ided for the introd ction of the amino or thiogro ps at ario s positions in the oligon cleotides, for the attachment ofario s reporter gro ps and for the p rification of oligon cleotide

conj gtes . The posts nthetic f nctionali ation of oligon cleotides forthe introd ction of reporter gro ps is disc ssed

V

i Preface

in Chapter 5 . Protocols for the se of oligon cleotide conj gates asdiagnostic probes are described in Chapter 6 .

In Chapter 7, the a thors ha e incorporated t o ne degen-erate bases into oligon cleotides and ha e disc ssed their ses. Verrecentl , the se of oligon cleotides containing degenerate bases hasbeen increasing in the area of pol merase chain reaction. Chapter 8describes protocols for the incorporation of radio-labeled n cleotidesinto oligon cleotides . The remaining chapters (Chapters 9-14)describe ario s anal tical techniq es and their applications to oli-gon cleotides, oligon cleotide analogs, and oligon cleotide conj -gates. The techniq es described in the present ol me are highperformance liq id chromatograph (Chapter 9), seq encing (Chap-ter 10), gel-capillar electrophoresis (Chapter 11), n clear magneticresonance (Chapter 12), mass spectrometr (Chapter 13), and thermo-d namic stabilit (Chapter 14) .

I o ld like to thank all the a thors of ario s chapters of thisbook and all the credit for the book's teachings goes to them . I amalso thankf l to Pa l Zamecnik, Thor Pederson, and Dan Bro n forfr itf l s ggestions . I o ld also like to thank John Walker, Meth-ods in Molec lar Biolog series editor, for his g idance at all stagesof editing . Finall , man thanks go to Thomas Lanigan and FranLipton of H mana Press, hose hard ork and diligence ha e madethis book a realit .

S dhir Agra alWorcester Fo ndation for E perimental Biolog

222 Maple A en eShre sb r , MA 01545

Present Address :H bridon Inc .

One Inno ation Dri eWorcester, MA 01605

Fore ord

Since the disco er of the n cleic acids in the latter part of the lastcent r, se eral decades elapsed before the str ct res of the constit entp rines and p rimidines, the corresponding n cleosides, and the nat re ofthe intern cleotide bonds in the pol n cleotides ere clarified . B 1952,the chemical str ct res of both classes of n cleic acids, RNA and DNA,had been established . Soon follo ed the Watson-Crick proposal for theDNA str ct re and this shered in a ne era in chemical, biochemical, andmolec lar biological st dies .

As ith other classes of biological macromolec les, interest in thes nthesis of oligo- and pol n cleotides increased er rapidl and s s-tematic st dies on the s nthesis of pol n cleotides ere ndertaken in themid- 1950s . Indeed, strategies ere s ccessf ll de eloped for the s n-thesis of oligon cleotides of defined seq ences. These, in conj nction

ith en matic approaches enabled the s nthesis of high molec lar eightpol mers that ere s ccessf ll sed in the el cidation of the geneticcode and later in the total laborator s nthesis of genes f ll f nctionalin i o .

In more recent ears ne de elopments in s nthesis ha e ad ancedthe s nthetic technolog to a highl efficient le el. In partic lar, s nthe-sis on pol mer s pports, a concept first de eloped b Merrifield inpol peptide s nthesis, has res lted in enormo s impro ements and speedin s nthesis of large si e pol n cleotides .

Dr. Agra al is to be congrat lated on ha ing prod ced t o a thori-tati e books ith contrib tions from e perts in different aspects of pol -n cleotide s nthesis. These monographs sho ld be e tremel sef l tothose ho are alread practitioners in the field and also those ho antto enter this field itho t an partic lar backgro nd.

H. Gobind KhoranaMassach setts Instit te of Technolog

Cambridge, MA

ii

Contents

PrefaceFore ord iiCompanion Vol me Contents iContrib tors iii

i

CH . 1 . Protecting Gro ps in Oligon cleotide S nthesis,Etienne Son ea 1

CH. 2. Incorporation of Modified Bases into Oligon cleotides,Rich B. Me er, Jr 73

CH. 3 . F nctionali ation of Oligon cleotides ith Amino Gro psand Attachment of Amino Specific Reporter Gro ps,

S dhir Agra al 93CH. 4 . F nctionali ation of Oligon cleotides

b the Incorporation of Thio-Specific Reporter Gro ps,Jacq eline A . Fidan a, Hiroaki O aki, and Larr W. McLa ghlin . . . 121

CH. 5 . Posts nthesis F nctionali ation of Oligon cleotides,Barbara C. F. Ch and Leslie E. Orgel145

CH. 6 . Oligon cleotide-En me Conj gates,Jerr L. R th 167

CH. 7 . Oligon cleotides Containing Degenerate Bases :S nthesis and Uses,

Pa l Kong Thoo Lin, and DanielM. Bro n187CH. 8 . S nthesis of [' 5N]-Labeled DNA Fragments,

Roger A. Jones 207CH. 9 . Anal sis and P rification of S nthetic Oligon cleotides

b High-Performance Liq id Chromatograph ,William J. Warren and George Vella 233

CH. 10 . Seq ence Anal sis of Oligodeo ribon cleotides,Diane M. Black and Peter T. Gilham 265

CH. 11 . Gel-Capillar Electrophoresis Anal sis of Oligon cleotides,Ale Andr s 277

Contents

CH . 12 . N clear Magnetic Resonance St dies of Oligon cleotides,Jer W. Jaros e sk4 Siddhartha Ro , and Jack S. Cohen301

CH. 13. Mass Spectrometr of N cleotides and Oligon cleotides,Thomas D. McCl re and Karl H. Schram319

CH . 14 . E tracting Thermod namic Data from Eq ilibri m Melting C r es forOligon cleotide Order-Disclos re Transitions,

Kenneth J. Bresla er 347

Inde 373

Contents of the companion ol me:

Protocols for Oligon cleotides and AnalogsS nthesis and Properties

CH . 1 . A Brief Histor of Oligon cleotide S nthesis,Daniel M. Bro n

CH . 2 . Oligon cleotide S nthesis : Phosphotriester Approach,Chris Christodo lo

CH . 3 . Oligodeo ribon cleotide S nthesis : Phosphoramidite Approach,Serge L. Bea cage

CH . 4 . Oligodeo n cleotide S nthesis : H-Phosphonate Approach,Brian C. Froehler

CH . 5 . Oligoribon cleotide S nthesis : The Sil l-Phosphoramidite Method,Masad J. Damha and Kel in K Ogil ie

CH . 6 . S nthesis of 2'-O-Alk loligoribon cleotides,Brian S. Sproat

CH . 7 . An Impro ed Method for the S nthesis and Deprotectionof Meth lphosphonate Oligon cleotides,

Richard I. Hogrefe, Mark A . Re nolds, Morte a M. Vaghefi, Ke in M.Yo ng, Timoth A. Rile , Robert E. Klemand L le J. Arnold, Jr.

CH . 8 . Oligon cleoside Phosphorothioates,Gerald Zon

CH . 9 . S nthesis and P rification of Phosphorodithioate DNA,W. T. Wiesler, W. S. Marshall, and M. H. Car thers

CH . 10. Oligodeo ribon cleotide Phosphotriesters,Maria Ko iolkie ic and Andr ej Wilk

CH . 11 . Oligon cleoside Boranophosphate (Borane Phosphonate),Barbara Ramsa Sha , Jon Madison, An p Sood,

and Bernard F. Spiel ogelCH . 12. Oligon cleotide Phosphorofl oridates and Fl oridites

Wojciech Dabko ski, Jan Michalski, and Friedrich CramerCH . 13 . a-Oligodeo n cleotides,

Franfois Mor an, Bernard Ra ner, and Jean-Lo is ImbachCH . 14 . Stereospecific S nthesis of P-Chiral Analogs of Oligon cleotides,

Wojciech J. Stec and Zbignie J. Lesniko skiCH. 15 . Oligon cleotide Analogs ith Dimeth lenes lfide, -s lfo ide,

and -s lfone Gro ps Replacing Phosphodiester Linkages,Zhen H ang, K Christian Schneider, and Ste en A. Benner

CH. 16 . Oligon cleotide Analogs Containing Dephospho-Intem cleoside Linkages,E gen Uhlmann and An sch Pe man

CH . 17 . Scale-Up of Oligon cleotide S nthesis : Sol tion Phase,H. Seliger

CH. 18 . Large-Scale Oligon cleotide S nthesis Using the Solid-Phase Approach,Nanda D. Sinha

CH . 19 . Solid-Phase S pports for Oligon cleotide S nthesis,Richard T. Pon

Contrib tors

SUDHIR AGRAWAL Worcester Fo ndation for E perimental Biolog ,Shre sb r, MA

ALEX ANDRUS Applied Bios stems, Inc ., Foster Cit , CADIANE M. BLACK Instit t de Chimie Biologiq e, Fac lte de Medecine,

Strasbo rg, FranceKENNETH J. BRESLAUER Department of Chemistr, R tgers, The State

Uni ersit of Ne Jerse , Piscata a , NJDANIEL M. BROWN Laborator of Molec lar Biolog , MRC, Cambridge, UKBARBARA C. F. CHU The Salk Instit te for Biological Sciences, San Diego, CAJACK S. COHEN Department of Pharmacolog , Georgeto n Uni ersit,

Rock ille, MDJACQUELINE A. FIDANZA Department of Chemistr, Boston College, Boston, MAPETER T. GILHAM Department of Biological Sciences, P rd e Uni ersit,

West Lafa ette, INJERZY W. JAROSZEWSKI Department of Organic Chemistr, Ro al Danish

School of Pharmac , Copenhagen, DenmarkROGER A. JONES Department of Chemistr, R tgers, The State Uni ersit

of Ne Jerse , Piscatat a , NJPAUL KONG THOO LIN Laborator of Molec lar Biolog , MRC, Cambridge, UKTHOMAS D. MCCLURE Department of Pharmace tical Sciences, The

Uni ersit of Ari ona Health Sciences Center, T cson, AZLARRY W. McLAUGI-ILIN Department of Chemistr, Boston College, Boston, MARICH B. MEYER, JR . MicroProbe Corporation, Bothell, WALESLIE E. ORGEL The Salk Instit te for Biological Sciences, San Diego, CAHIROAKI OZAKI Department of Chemistr, Boston College, Boston, MASIDDHARTHA Ro Department of Bioph sics, Bose Instit te, Calc tta,

IndiaJERRY L. RUTH US Fish and Wildlife Forensics Laborator , Ashland, OR

att

i Contrib tors

KARL H. SciRA i Department of Pharmace tical Sciences, The Uni er-sit of Ari ona Health Sciences Center, T cson, AZ

ETIENNE SONVEAUX Laboratoire de Biochimie Ph siq e et desBiopol meres, Lo ain-La-Ne e, Belgi m

GEORGE VELLA Millipore Corporation, Waters Chromatograph Di ision,Milford, MA

WILLIAM WARREN Millipore Corporation, Waters ChromatographDi ision, Milford, MA