New NTP analogs: the synthesis of 4′-thioUTP and 4′-thioCTP and their utility for SELEX

Abstract

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′-NH2dUTP and 2′-NH2dCTP; 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).

Functional Aptamers and Aptazymes in Biotechnology, Diagnostics, and Therapy

“…Other potential sites for chemical modification that may be beneficial for the properties of therapeutic aptamers are the purine and pyrimidine residues, 162-175 sugars, [176][177][178][179][180] and phosphate residues, 181 which all have been shown to be compatible with template-directed enzymatic incorporation into DNA and with all necessary steps required for a complete in Vitro selection cycle. 175,182 In general, modified nucleotides are increasingly being utilized in all categories of therapeutic oligonucleotides to increase nuclease resistance, target affinity, and specificity.…”

Improving the Stability of Aptamers by Chemical Modification

“…The partially 4'-thio-modified "thioRNA5" exhibited 3-fold stronger nuclease resistance than the unmodified aptamer, and had a higher thermostability with the melting temperature around 45 o C. On the contrary, the original aptamer had a melting temperature only around 35 o C [75]. Later on, Kato et al [76] found that given a leader sequence of about +15 of DNA template, the 4'-thioUTP and 4'-thioCTP could be successfully incorporated by the T7 RNA polymerase with higher efficiency than traditional 2'-fluoride and 2'-amino modifications. The 59mer thio-RNA synthesized in this way had a halflife around 1174 minutes towards RNase A, about 50 times greater than the unmodified RNA, though its stability was weaker than the traditional 59-mer 2'-NH 2 -RNA which completely resisted the RnaseA attack.…”

“…The 59mer thio-RNA synthesized in this way had a halflife around 1174 minutes towards RNase A, about 50 times greater than the unmodified RNA, though its stability was weaker than the traditional 59-mer 2'-NH 2 -RNA which completely resisted the RnaseA attack. In another example, the direct SELEX versus human -thrombin was able to generate a 4'-thio-pyrimidine RNA aptamer with high affinity, though its stability was not disclosed [53] [76]. Matsuda et al found that RNA aptamers with 4'-thioribonucleosides can be 1100 times more stable than normal RNA in 50% human plasma.…”

Aptamers: Ligands For All Reasons

“…Similarly, ribonucleoside boranophosphates have been demonstrated to be incorporated by T7 RNA polymerase (Shaw et al, 2003). This enzyme is also able to polymerize transcripts containing 4 -thiopyrimidines ( Figure 1.2a), a modification that increases their stability by about 50-fold relative to unmodified RNA (Kato et al, 2005). It was recently reported that the combination of mutated T7 RNA polymerases, Y639F and Y639F/H784A, allows the efficient incorporation of all four 2 -O-methyl nucleotides (Chelliserrykattil and Ellington, 2004;Burmeister et al, 2005Burmeister et al, , 2006.…”