Enzymatic Primer-Extension with Glycerol-Nucleoside Triphosphates on DNA Templates 英文参考文献.docVIP
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Enzymatic Primer-Extension with Glycerol-Nucleoside Triphosphates on DNA Templates 英文参考文献
EnzymaticPrimer-ExtensionwithGlycerol-Nucleoside
TriphosphatesonDNATemplates
JesseJ.Chen1.,Ching-HsuanTsai1.¤,XinCai1,AllenT.Horhota2,LarryW.McLaughlin2,JackW.
Szostak1*
1HowardHughesMedicalInstitute,andDepartmentofMolecularBiologyandCenterforComputationalandIntegrativeBiology,MassachusettsGeneralHospital,Boston,
Massachusetts,UnitedStatesofAmerica,2DepartmentofChemistry,MerkertChemistryCenter,BostonCollege,ChestnutHill,Massachusetts,UnitedStatesofAmerica
Abstract
Background:Glycerolnucleicacid(GNA)hasanacyclicphosphoglycerolbackbonerepeat-unit,butformsstableduplexes
based on Watson-Crick base-pairing. Because of its structural simplicity, GNAis of particular interest with respect to the
possibilityofevolvingfunctionalpolymersbyinvitroselection.Template-dependentGNAsynthesisisessentialtoanyGNA-
basedselectionsystem.
Principal Findings: In this study, we investigated the ability of various DNA polymerases to use glycerol-nucleoside
triphosphates (gNTPs) as substrates for GNA synthesis on DNA templates. Therminator DNA polymerase catalyzes
quantitativeprimer-extensionbytheincorporationoftwoglyceronucleotides,withmuchlessefficientextensionuptofive
glyceronucleotides.Steady-statekineticexperimentssuggestedthatGNAsynthesisbyTherminatorwasaffectedbyboth
decreasedcatalyticratesandweakenedsubstratebinding,especiallyforpyrimidines.Inanattempttoimprovepyrimidine
incorporation by providing additional stacking interactions, we synthesized two new gNTP analogs with 5-propynyl
substitutedpyrimidinenucleobases.ThisledtomoreefficientincorporationofgC,butnotgT.
Conclusions:WesuggestthatdirectedevolutionofTherminatormightleadtomutantswithimprovedsubstratebinding
andcatalyticefficiency.
Citation: Chen JJ, Tsai C-H, Cai X, Horhota AT, McLaughlin LW, et al. (2009) Enzymatic Primer-Extension with Glycerol-Nucleoside Triphosphates on DNA
Templates.PLoSONE4(3):e4949.doi:10.1371/journal.pone.0004949
Editor:FloydRomesberg,TheScrippsResearchInstitute,UnitedStatesofAmerica
ReceivedFebruary2,
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