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 alpha-thrombin using thioUTP and thioCTP, and found a thioRNA aptamer with high binding affinity (K(d) = 4.7 nM).

Scheme 1

(a) MMTrCl, pyridine then Ac₂O, DMAP; (b) 80% aq. AcOH; (c) 2-chloro-4H-1,3,2-benzodioxaphosphorin-4-one, pyridine–DMF; (d) bis(tri-n-butylammonium)pyrophosphate in DMF, Bu₃N; (e) 1% iodine in pyridine–H₂O then NH₄OH.

Figure 1

Sequences of DNA templates and the resulting RNAs by in vitro transcription. The italic regions were T7 promoter sequence.

Figure 2

PAGE analysis of in vitro transcription by T7 RNA polymerase using template1. In vitro transcription was carried out as described in Materials and Methods in the presence of GTP (lane 1); GTP and ATP (lane 2); GTP, ATP and CTP (lane 3); four NTPs (lane 4); GTP, ATP and CTP plus thioUTP (lane 5); GTP, ATP and UTP plus thioCTP (lane 6); and GTP and ATP plus thioUTP and thioCTP (lane 7).

Figure 3

PAGE analysis of in vitro transcription by T7 RNA polymerase using template2. In vitro transcription was carried out as described in Materials and Methods in the presence of four NTPs (lanes 3–6, for 10, 30, 60 and 180 min, respectively); GTP and ATP plus thioUTP and thioCTP (lanes 7–10, for 10, 30, 60 and 180 min, respectively); GTP and ATP plus 2′-NH₂dUTP and 2′-NH₂dCTP (lanes 11–14, for 10, 30, 60 and 180 min, respectively), and GTP and ATP plus 2′-FdUTP and 2′-FdCTP (lanes 15–18, for 10, 30, 60 and 180 min, respectively). Lanes 1 and 2 are sequence marker obtained by treatment of RNA30 with 50 mM aq. Na₂CO₃ (pH 9.0) and RNase T1, respectively.

Figure 4

Time course of transcription efficiency. The amount of full-length RNAs is presented in % compared with the amount of RNA30 (100%) after 360 min.

Figure 5

(A) PAGE analysis of reverse transcription by Superscript™ II (RNase H⁻). Reverse transcription was carried out as described in Materials and Methods in the presence of labeled primer only (lane 1); labeled primer and RNA59 as a template (lane 2); labeled primer and thioRNA59 as a template (lane 3). (B) Electropherogram of DNA-sequencing fragments.

Figure 6

Stabilities of RNA59, thioRNA59 and aminoRNA59 toward RNase A. (A) PAGE analysis of labeled RNAs incubated at 37°C in the presence of RNase A. (B) Time course of degradation of RNAs by RNase A.

Figure 7

Sequences identified in the 10th round thioRNA library derived from the affinity selection performed on human α-thrombin. The frequency of clones carrying the same sequence is indicated in parenthesis. The conserved sequences in each individual class are indicated by back ground colors. The sequences common to all ligands are shown in lower case letters and the sequences of the variable region in upper case letters.

Figure 8

The secondary structures of DNA-G4, RNA-24 and thioRNA aptamers, CI-2-23 and CII-1-37.

Figure 9

The binding of DNA-G4, RNA-24, Cl-2-23 and CII-1-37 to varying concentration of human α-thrombin was determined by nitrocellulose filter partitioning as described in Materials and Methods.