new ntp analogs the synthesis of 4thioutp and 4thioctp and their utility for selex the synthesis of the triphosphates of 4thiouridine and 4thiocytidine 4thioutp 7 thioutp and 4thioctp 8 thioctp and their utility for selex systematic evolution of ligands by exponential enrichment is described the new nucleoside triphosphate ntp analogs 7 and 8 were prepared from appropriately protected 4thiouridine and cytidine derivatives using the onepot method reported by j ludwig and f eckstein 1989 j org chem 54 631635 because selex requires both in vitro transcription and reverse transcription we examined the ability of 7 and 8 for selex by focusing on the two steps incorporation of 7 and 8 by t7 rna polymerase to give 4thiorna thiorna proceeded well and was superior to those of the two sets of frequently used modified ntp analogs for selex 2nh2dutp and 2nh2dctp 2fdutp and 2fdctp when an adequate leader sequence of dna template was selected we revealed that a leader sequence of about 15 of dna template is important for the effective incorporation of modified ntp analogs by t7 rna polymerase in addition reverse transcription of the resulting thiorna into the complementary dna in the presence of 2deoxynucleoside triphosphates dntps also proceeded smoothly and precisely the stability of the thiorna toward rnase a was 50 times greater than that of the corresponding natural rna with these successful results in hand we attempted the selection of thiorna aptamers to human alphathrombin using thioutp and thioctp and found a thiorna aptamer with high binding affinity kd 47 nm