The synthesis of the triphosphates of 4′-thiouridine and 4′-thiocytidine, 4′-thioUTP (7; thioUTP) and 4′-thioCTP (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 4′-thiouridine and -cytidine derivatives using the one-pot method reported by J. Ludwig and F. Eckstein [(1989) J. Org. Chem ., 54, 631–635]. 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 4′-thioRNA (thioRNA) proceeded well and was superior to those of the two sets of frequently used modified NTP analogs for SELEX (2′-NH 2 dUTP and 2′-NH 2 dCTP; 2′-FdUTP and 2′-FdCTP), 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 2′-deoxynucleoside 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 α-thrombin using thioUTP and thioCTP, and found a thioRNA aptamer with high binding affinity ( Kd = 4.7 nM).

A monoclonal antibody (TDM-2) specific to a UV-induced cyclobutane pyrimidine dimer (T[c/s-syn]T) has previously been established; however, the immunization had used UV-irradiated calf-thymus DNA containing a heterogeneous mixture of photoproduct sites. We investigated here the structural requirements of antigen recognition by the antibody using chemically synthesized antigen analogs. TDM-2 bound with cis-syn, but not trans-syn thymine dimer, and could bind strongly with four nucleotide analogs in which the cis-syn pyrimidine dimer was located in the center. Antigen analogs containing abasic linkers at the 5′- or 3′-side of the cis-syn cyclobutane pyrimidine dimer were synthesized and tested for binding to TDM-2. The results indicated that TDM-2 recognizes not only the cyclobutane ring but also both the 5′- and 3′-side nucleosides of the cyclobutane dimer. Furthermore, it was proved that either the 5′- or 3′-side phosphate group at a cyclobutane dimer site was absolutely required for the affinity to TDM-2. The antibody showed a strong binding to single stranded DNA but indicated little binding to double stranded DNA.