Free-Energy Landscape of a Thrombin-Binding DNA Aptamer in Aqueous Environment

Abstract

Thrombin-bindingaptamer(TBA-15)isasingle-stranded15-meroligonucleotidethathasawiderangeof biomedicalapplications.Inthepresenceofmetalcationsofpropersizes,thisaptamerdisplaysG-quadruplexeswithasingle cationenclosedatitscentralbindingsitewhenitiscompletelyfolded.Tounderstandhowthisaptamerfoldsintoitsstablethreedimensional structureinthepresenceofK+ions,wecarriedoutfree-energycalculationsusingthestate-of-artreplicaexchange moleculardynamicssimulation(REMD)attheall-atomlevel.TheresultingfreeenergymaprevealedthatTBA-15followsatwostate foldingbehaviorwithasubstantiallylargefoldingbarrierof6kcal/molatambienttemperature.Oursimulationshowedthat theinterveningTGT-loop,whichislocatedinthemiddleoftheTBA-15sequence,virtuallyremainsintactregardlessoffolding andunfoldingstates.Furthermore,intheconservedTGT-loopstructure,thebase-pairstackingofG8andT9inducesthenativelike baseorientationsofG6andG10pertainingtotheupperG-quadrant.Thisstackinginteractionenhancestheloopstability andreducesitsdynamicfluctuations.Interestingly,fortheG-stemtofoldintoitsnativestate,theaggregationoftheG8andT9 residuesintheTGT-loopisakeystepforinitiatingthefoldingeventoftheG-stembycapturingabulkycation.