Alternative nucleic acid : the expanded genetic alphabet

195 Pages·1999·7.7 MB·English

by  Lutz, Stefan, 1969-

ALTERNATIVENUCLEICACID:
THEEXPANDEDGENETICALPHABET
By
STEEANLUTZ
ADISSERTATIONPRESENTEDTOTHEGRADUATESCHOOLOFTHE
UNIVERSITYOFFLORIDAINPARTIALFULFILLMENTOFTHEREQUIREMENTS
FORTHEDEGREEOFDOCTOROFPHILOSOPHY
UNIVERSITYOFFLORIDA
1999
Copyright1999
by
StefanLutz
Dedicatedtomyfamily
fortheirendlessloveandsupport
ACKNOWLEDGMENTS
Firstandforemost,IwouldliketothankDr.StevenA.Bennerfortheopportunityto
workinhislab.Withouthissupportandenthusiasm,thisworkwouldnothavebeenpossible.
MythanksgotothepastandpresentmembersofthenonstandardBennerresearch
group,includingDr.PetraBurgstaller,Dr.TomBattersby,Dr.KevinDevine,HeikeHeld,
Dr.MichaelLutz,DarwinAng,MikeThompson,MatCarrigan,VicOhtamaa,Dr.Janos
Kodra,Dr.JeongHoPark,MikeSismour,Dr.SimonaJurczyk,andmysuccessor,Cynthia
Whitehead.Aspecialthanksgoestomytwoundergraduateresearchers,KenParkandKim
Ferguson,foralltheirinputintothisresearch.
Forthesupplyofprecursormaterial,IwouldliketoacknowledgeAndreMueller
(ETH,Zurich;2,4-diamino5-ribofuranosyl-pyrimidine)andDr.SimonaJurczyk(Sulfonics,
Eragen,AlachuaEL)forthe2’-deoxyxanthosinephosphoramidite.Forexperimentsinvolving
thetriphosphateofpyDAD,thematerialpreparedbyJenHorlacherwasgreatlyappreciated.
Dr.JeongHoParkandMikeSismourkindlyprovidedtheHIVtype-1reversetranscriptase
fortheincorporationstudiesdiscussedinChapter3.
IamalsoverygratefultoYvesChoffat(ZoologischesInstitut,UniveristyofZurich),
Jace Dinehart (ICBRDNAsynthesiscorefacility. UniversityofFlorida), andDr. Tom
Battersbyfortheextraeffortinsynthesizingtheoligonucleotides;containingthenonstandard
nucleotides.
IV
Furthermore, I wish to thank ourcollaborators: Dr. G. Folkers (ETH Zurich;
phosphorylationofnonstandardnucleotides byHerpesSimplexVirustype-1 thymidine
kinase),Dr.MaryKopka(DickersonLaboratory,UCLA;crystallizationofoligonucleotides
containingnonstandardbasepairs),andthefolksatNewEnglandBiolabs (Beverly,MA;
supportincloningandexpressionofthermostableDNApolymerases).
Last,butnotleast,Iwantexpressmythankstoallthepoorfellowswhoactually
volunteeredtoreadandcorrectthisworkatonestageoranother:Dr.PetraBurgstaller,Dr.
Kevin G. Devine, Dr. Tom Battersby, Dr. DavidLiberies, LisaManning, andJennifer
Cottone.
TABLEOFCONTENTS
ACKNOWLEDGMENTS iv
SYMBOLSANDABREVIATIONS ix
ABSTRACT xiii
CHAPTERS
INTRODUCTION
1 1
RibozymesandDeoxyribozymes-NovelCatalystsfromNucleicAcid 2
InVitroSelection/SELEX 3
InVitroEvolutionofNucleicAcid 4
TheSecretsofNucleicAcidCatalysis 6
TheNextGenerationNucleicAcidEnzymes 8
ExpandingtheGeneticAlphabet 13
ResearchObjectives 16
2 THECHEMISTRYOFALTERNATIVENUCLEICACID 17
Introduction 17
SyntheticApproachesforpyDADandpuADA 18
ThepuADAnucleoside 18
ThepyDADnucleoside 18
StructuralfeaturesofthepyDADnucleoside 20
StructuralEffectsofpyDADandpuADAinOligonucleotides 20
Meltingstudiesofoligonucleotides 22
MaterialsandMethods 23
ExperimentalProcedure 23
ResultsandDiscussion 35
TheChemistryofpyDADandpuADA 35
2’-Deoxyxanthosine:thepuADAnucleoside 35
VI
2,4-Diamino-5-(2’-deoxyribofuranosyl)-pyrimidine:thepyDADnucleoside 36
Structuralstudieson2,4-diamino-5-(2’-deoxyribofuranosyl)-pyrimidine 39
ThepyDAD/puADANonstandardBasePairinOligonucleotides 41
Meltingstudiesofoligonucleotides 43
Discussion 50
Conclusions 52
3 INCORPORATIONOFNONSTANDARDNUCLEOTIDESBYHIV-1REVERSE
TRANSCRIPTASE 54
Introduction 54
HIVType-1ReverseTranscriptase 55
HIVreversetranscriptasemutantsandnonstandardbasepairs 57
HIVreversetranscriptasemutantAZT-21andM184V 58
HIVreversetranscriptasemutantY181IandY188L 60
MaterialandMethods 61
ExperimentalProcedure 61
ResultsandDiscussion 66
IncorporationofpyDADandpuADAbyHIVReverseTranscriptase 66
Primerextensionexperiments 67
Primerextensionovertworounds 69
Standingstartexperiments 72
PCRAmplificationwithHIVReverseTranscriptase 75
DNASequencingwithNonstandardNucleotides 77
Conclusions 78
4 PCRWITHPSEUDOTHYMIDINE:HIGHCAPACITYSCREENINGOFDNA
POLYMERASESUSINGTHESCINTILLATIONPROXIMITYASSAY 82
Introduction 82
StructuralEffectsofC-NucleosidesonPolymerasePerformance 84
C-Nucleosidesvs.N-Nucleosides 84
High-ThroughputScreenforDNAPolymerases 86
ScintillationProximityAssay 87
MaterialsandMethods 88
ExperimentalProcedure-ChemicalSynthesis 88
ExperimentalProcedure-MolecularBiology 92
ResultsandDiscussion 95
InVitroScreeningAssay 95
Synthesisofpseudothymidine 96
Primerextensionexperiments 96
Standingstartexperiments 99
Steady-statekineticsof\j/T-incorporationbygelfidelityassay 101
PCRamplification 102
vii
Amplificationfidelityandsequencing 104
Conclusions 107
5 TOWARDSDIRECTEDEVOLUTIONOFDNAPOLYMERASESTHAT
RECOGNIZEANEXPANDEDGENETICALPHABET 109
Introduction 109
DirectedEvolutionofEnzymes 110
MutationStrategies Ill
MutationstrategyforDNApolymerases 113
FunctionalProteinExpressionandMicrobialHosts 114
TheBL21(DE3)pLysS/pETexpressionsystem 114
ExpressionofarchaeaproteininE.coli 115
ExpressionvectorpAlllZandpSBETa 117
High-ThroughputScreeningAssays 117
MaterialsandMethods 119
Experimentalprocedure 119
ResultsandDiscussion 125
Exonuclease-DeficientPfuDNAPolymerasebyOverlappingPCR 125
DNAShuffling 128
ProteinExpressionSystemforDNAPolymerases 131
Ligationofthegenelibrary 131
Expressionvectorandhostorganism 132
Proteinexpressionlibraries 135
PurificationofoverexpressedDNApolymerases 136
DNAShufflingandProteinExpressionofVentandPfuDNAPolymerases 137
SPAwiththepyDAD/puADABasePair 138
PreliminarySPAwithHIVreversetranscriptase(Y188L) 138
SPAwiththepSBETa-9°Nlibrary-design 142
SPAwiththepSBETa-9°Nlibrary-experimentaldata 144
Conclusions 149
APPENDICES
A GENETICCODESANDAMINOACIDSCODES 153
B MATERIALS 154
C GENERALMETHODSINMOLECULARBIOLOGY 160
REFERENCES 163
BIOGRAPHICALSKETCH 179
viii
SYMBOLSANDABREVIATIONS
A adenosine
AIBN 2,2'-azobisisobutyronitrile
AIDS acquiredimmunodeficiencysyndrome
Amp ampicillin
AMV avianmyeloblastosisvirus
arom aromatic
APS ammoniumpersulfate
ATP adenosinetriphosphate
Bn benzyl
bp basepair(s)
BSA bis(trimethylsilyl)acetamide
C cytidine
Ci Curie(1Ci=3.7x10^Bequerel)
CM chloramphenicol
COSY 2-dimensionalcorrelationspectroscopy
CPG controlledporeglass
cpm countsperminute
d days
Da Dalton
ddN dideoxynucleoside(ddA,ddC,ddG,ddT)
IX
dN deoxynucleoside (dA,dG,dC,\|/T,d\|/U,d(pyDAD),d(puADA))
DBU 1.8-diazabicyclo[5.4.0]undec-7-ene
DMAP 4-dimethylaminopyridine
DMF N,N-dimethylformamide
DMSO dimethylsulfoxide
DMT 4',4"-dimethoxytrityl
DTT 1,4-dithio-DL-threitol
£ absorptioncoefficient(M'crn')
E.coli Escherichiacoli
EDTA ethylendiaminotetraacetate
ESI electrosprayionization
EtOH ethanol
FAB fastatombombardment
G guanosine
h hours
HIV-1 humanimmunodeficiencyvirustype-1
HMDS hexamethylendisilazane
HPLC highperformancehquidchromatography
HV highvacuum
IPTG isopropyl-P-D-thiogalactopyranoside
IRR irritationradiation(NOE)
Kan kanamycin
wavelength(nm)
LB Luria-Bertanimedium