Searching molecular landscapes for the evolution of primal catalysts : in vitro selection of DNA-based ribonucleases

198 Pages·2002·8.3 MB·English

by  Carrigan, Matthew A.

SEARCHINGMOLECULARLANDSCAPESFORTHEEVOLUTIONOFPRIMAL
CATALYSTS: INVITROSELECTIONOFDNA-BASEDRIBONUCLEASES
By
MATTHEWA.CARRIGAN
DISSERTATIONPRESENTEDTOTHEGRADUATESCHOOLOFTHE
UNIVERSITYOFFLORIDAINPARTIALFULFILLMENTOFTHE
REQUIREMENTSFORTHEDEGREEOFDOCTOROFPHILOSOPHY
UNIVERSITYOFFLORIDA
2002
Thisworkisdedicatedtomyson,Christian.
11
ACKNOWLEDGMENTS
Iwouldliketothankmyparentsandfamilyfortheircontinuoussupport. Iwould
liketothankseveralpeoplefortheirearlyencouragementinmycareerinscience,
specificallymyfather,aswellasBillCrabtree,TracyBailey,KenCline,RalphHenry,
andMikeMcCaffery. IamindebtedtothescientificcollaborationofDr.Maury
Swansonandhisentirelab,specificallyCarlUrbinati,RonHector,andKeithNykamp.
AlonsoRicardohelpedmetremendouslytoexecutemanyoftheexperimentsdescribedin
thisdissertation. MikeThomsonofferedinvaluableandunendingassistance. Without
StevenBenner'spatienceandenthusiasmforscience,thisresearchendeavorwouldnot
havebeenpossible.
in
1
TABLEOFCONTENTS
ACKNOWLEDGEMENTS
iii
ABBREVIATIONS vii
ABSTRACT ix
CHAPTER page
1 INTRODUCTION 1
Requirementsofa"Living"System 1
ChemicalChallengestothePrebioticSynthesisof"Life" 4
ProgressTowardsUnderstandingtheGenesisofLife:
DualFunctionBiopolymers 6
ProgressTowardsUnderstandingtheGenesisofLife:
PrebioticSynthesisofPotentiallyUsefulMonomersandPolymers 8
TheNextChallenge: ObtainingCatalyticFunctionfromRandomLibraries 1
ExperimentalDesign 19
2 MATERIALSANDMETHODS 24
PreparationofPrecursorDNAymesviaPCR 24
PreparationofSingle-StrandedDNAzymes 26
5'-EndLabelingofDNA 28
DNAzymeKineticAssays 28
CloningandSequencingDNAzymes 29
InVitroSelection 31
3 RESULTSOF614ANALYSIS 36
ResearchObjectives 36
DevelopingMethodsforPreparingSingle-strandedDNAzymesby
ExonucleaseDegradationof5'-PhosphorylatedComplementaryStrand 37
AsymmetricPCRwith"Tails,"FollowedbyGelPurification 39
DNAzyme614withuncaged-andcaged-ribose 41
Achievingmaximaldeprotectionofcagedribose 41
Kineticprofileof614withuncaged-andcaged-ribose 42
IV
LaserDoesNotDamageRibose-614 44
DNAzyme614isNaCl-dependent,andMgCh-independent 45
UnderstandingReasonsforIncompleteCleavageofDNAzyme614 46
IncompleteDe-protectionofCaged-Ribose 46
IncompleteRemovaloftheComplementaryStrand 47
ApproachtoChemicalEquilibrium 48
AImrpertohpeer2l7y-Faonldd7e9d-nDuNcAlezoytimdeeF6r1a4gmentsActingasCatalystsorInhibitors?.5510
CloningandSequencingCleavedandUncleaved614Near
CleavagePlateau 54
DNAzyme614Cleavesincisandtrans 56
CleavageRateofRibose-614Varieswith614Concentration 58
Deoxyribose-614CleavesVariousRibose-Substrates 62
CompetitionStudiesofRibose-614Cleavage 63
d-614CleavesFasterintransThanincis: Single-turnoverKinetics 64
d-614CleaveswithMultiple-turnover 68
Testing614RateofAssociationandDisassociation 68
StructuralAnalysisof614 74
SummaryofResults 81
4 STUDIESOFINVITROSELECTIONS 123
InVitroSelectionofFunctionalizedandNon-FunctionalizedLibraries
UsingCaged-RiboseandLiquid-PhaseSelection 123
InVitroSelection 123
TestingKineticsfromRound8PoolsWithoutCaged-ribose 127
TestingforInadvertentSelectionforSusceptibilitytoLaser 127
Caged-riboseDoesNotInduceCleavageintrans 128
KineticAnalysisofRounds1-8Poolsfor"H: ang+ribose" 128
SequenceEvolutionDuringIVS 131
KineticAnalysisofRound8Clones 133
SimulationsofInVitroSelectionExperiments 134
SimulatingInVitroSelections 135
UsingSimulationstoDeterminetheImpactoftheLeakage(L)
ParameteronIVS 139
ExaminingtheImpactoftheSelectionTime(St) 145
ExaminingtheImpactoftheRateoftheFastestCatalysts(kfasles,) 146
ExaminingtheImpactoftheReactiveFraction 147
ComparingLinearlyandExponentiallyDistributedInitialPopulations 148
DecipheringtheDistributionFunctionforCatalysts 149
SummaryofResults 152
5 SUMMARY 169
CatalyticBehaviorofDNAzymesfromIVSisMoreComplex
thenPreviouslyAssumed 170
Caged-riboseisUsefulforKinetics,ButIncreasesLeakage
TooMuchforIVS 173
IVSIsolatesSequenceVariantsof614withWideRangeofCatalyticPower 174
SimulationsAllowEstimationofCatalystDistribution 176
NucleicAcidCatalysisandtheOriginsofLife 177
REFERENCES 179
BIOGRAPHICALSKETCH 184
VI
ABBREVIATIONS
IVS invitroselection
bya billionyearsago
SL Sizeofinitiallibrary
RF ReactiveFraction
NRF Non-reactiveFraction: NRF=SL*(\-RF)
k„ Intrinsicrateofmolecule"«": eachmemberoftheinitiallibrary,n,is
assignedanintrinsicrate,k„, whichrelatestoitsabilitytobecleaved
underselectionconditions. Theintrinsicrateissequence-dependentand
thereforeunchanging.
'Alifen Theintrinsicrateofmolecule"«"(k„)expressedintermsofhalf-life:
'Alifen=ln2/k„*siowest Therateconstantfortheslowestcatalystsinthe
library.
k/asiest Therateconstantofthefastestmoleculeinthelibrary.
St Selectiontime: theamountoftimeallowedforamoleculetocleaveitself
andthereforeachieve"survival."
morb Distributionfunctionforreactivemolecules. Twofunctionshavebeen
testedthusfar,alinearfunctionwithslope=m,andanexponential
functionwithdecayrate=b.
L Leakage: thefractionofthepoolthat"survives"aroundofselection
independentofStandintrinsicrateconstant.
vn
O
caged-ribose aribose-adenosinewiththe2'OHreplacedwithanortho-nitrobenzyl
protectinggroup
r-primer "ang+ribose"49-nucleotidesubstratecontainingribose
r-X oligonucleotide"X"withribose-adenosine
d-X oligonucleotide"X"withdeoxy-adenosine
A Deoxyadenosinetriphosphate
G Deoxyguanosinetriphosphate
C Deoxycytidinetriphosphate
T Deoxythymidinetriphosphate
E-base 5-(3-aminoallyl)-2'deoxyuridinetriphosphate(seestructurebelow)
O O O
—
"O—P o—P—o—P—
oI- oI- oI-
OH H
kcat(uni) chemicalstepforunimolecularcatalysis
ki(unj) rateoffoldingforunimolecularcatalysis
k_i(Uni) rateofunfoldingforunimolecularcatalysis
kcat(bi) chemicalstepforbimolecularcatalysis
ki(bi) rateofassociationforbimolecularcatalysis
k-i(bi) rateofdisassociationforbimolecularcatalysis
BimolecularKineticScheme:
E+S ^i(bi) ES <cat(bi) EP SE==rr--66114,4orr-dli-b66l1o42orr-primer
-i(bi)
UnimolecularKineticScheme:
o14unf0|ded -k1(uni) 614f0|ded *cat(un-i) Products
k-i(uni)
vin
AbstractofDissertationPresentedtotheGraduateSchool
oftheUniversityofFloridainPartialFulfillmentofthe
RequirementsfortheDegreeofDoctorofPhilosophy
SEARCHINGMOLECULARLANDSCAPESFORTHEEVOLUTIONOFPRIMAL
CATALYSTS: INVITROSELECTIONOFDNA-BASEDRIBONUCLEASES
By
MatthewA.Carrigan
December2002
Chair: StevenA.Benner
Cochair: ArtEdison
MajorDepartment: Neuroscience
Ourcurrentunderstandingoftheoriginsoflifesuggeststhatcatalyticnucleic
acidsmustarisefromrandompolymersofnucleicacids. Searchingrandomlibrariesof
nucleicacidpolymershasnotproducedtheabundanceandpowerofcatalysisbelieved
necessarytospawnlife. Further,theadditionofchemicalfunctionalitytorandom
librarieshasfailedtoimprovethecatalyticpotentialofrandomlibrariesasanticipated.
Thisapparentdiscrepancybetweenobservationandexpectationmaybe
attributabletoseveralshortcomingsoftheexperimentaldesign. Activecatalystsmaybe
lostduringthepreparationoflibraries,orduringtheenrichmentforcatalysts.
Furthermore,onlyincompletedescriptionshavebeenattemptedforany,focusingonlyon
thefastestcatalystsinthelibrary,ratherthancapturingcompletelythedistributionof
catalyticpower. Itisconceivablethataddedfunctionality,oralterationsofthelibraryor
IX
selectionconditions,candramaticallyaltertheabundanceanddistributionofcatalysts
withoutalteringtherateofthefastestcatalyst.
Thegoalofthisthesisistodevelopmethodsforestimatingthedistributionof
catalystswithinarandomlibraryofDNAsequence. Toaidinthedevelopmentof
realisticmodels,webeganwithanindepthanalysisofthecatalyticbehaviorofan
individualDNAzyme,614. Thisanalysisrevealedanumberofsurprisingfeaturesofthis
DNAzymethatmaybegeneralizedtootherDNAzymescreatedthroughinvitro
Selection(IVS). AlthoughselectedwithaprotocolbelievedtoenrichforMg-dependent
self-cleavingcatalysts,614cleavesbothincisandtransindependentlyofMg^. Therate
of/rans-cleavageatsubstratesaturationis6-foldhigherthantherateofm-cleavage.
Further,bothcis-andtrans-cleavageratesareenhancedattemperaturesbelow25°C(the
temperatureatwhichtheselectionswereperformed),suggestingthatabalanceisreached
betweenaslowerchemicalstep(k^uni)ork^bo)andamorestabilizedassociationstep
(ki(uni)orki(bi))atlowertemperatures.
Mutagenesisof614anditssubstrateswasperformedtolearnaboutthestructure
of614cleavingincisandtrans. Compensatorymutationsrevealedthatoneofthe
intermolecularhelixesformedbetween614anditssubstrateisidenticaltoan
intramolecularhelixformedinds-acting614. The/raws-folded614,however,hasan
additional4base-pairhelixnotfoundincz's-folded614,andthismaypartiallyexplainthe
fasterrateoftrans-cleavageatsaturation.
Wealsoaddressedthepotentiallossofcatalystsby"prematurecatalysis"during
thepreparationofDNAzymepoolsintheIVS. Purificationofsingle-stranded
DNAzymeswasaccomplishedusingasymmetricPCRorexonucleasedegradation,