Enhancing the catalytic repertoire of nucleic acids: a systematic study of linker length and rigidity

Abstract

The incorporation of potentially catalytic groups in DNA is of interest for the in vitro selection of novel deoxyribozymes. A series of 10 C5-modified analogues of 2'-deoxyuridine triphosphate have been synthesised that possess side chains of differing flexibility and bearing a primary amino or imidazole functionality. For each series of nucleotide analogues differing degrees of flexibility of the C5 side chain was achieved through the use of alkynyl, alkenyl and alkyl moieties. The imidazole function was conjugated to these C5-amino-modified nucleotides using either imidazole 4-acetic acid or imidazole 4-acrylic acid (urocanic acid). The substrate properties of the nucleotides (fully replacing dTTP) with TAQ polymerase during PCR have been investigated in order to evaluate their potential applications for in vitro selection experiments. 5-(3-Aminopropynyl)dUTP and 5-(E-3-aminopropenyl)dUTP and their imidazole 4-acetic acid- and urocanic acid-modified conjugates were found to be substrates. In contrast, C5-amino-modified dUTPs with alkane or Z-alkene linkers and their corresponding conjugates were not substrates. The incorporation of these analogues during PCR has been confirmed by inhibition of restriction enzyme digestion using XBAI and by mass spectrometry of the PCR products.

Figure 1

The synthesis of protected C5-amino-modified 2′-deoxyuridines from 5-iodo-2′-deoxyuridine. (i) N-propynyltrifluoroacetamide, Pd(PPh₃)₄, CuI, Et₃N, DMF, 60% yield; (ii) N-propenyltrifluoracetamide, Na₂PdCl₄, aq NaOAc, pH 5.2, 30–50% yield (23); (iii) NiCl₂/NaBH₄, MeOH, –78°C, 55% yield (for 1–3) (27); (iv) H₂, PtO₂ in MeOH, 1 h at room temperature, 88% yield.

Figure 2

The C5-modified triphosphates employed in this study. The triphosphates were prepared according to the procedures described in Materials and Methods.

Figure 3

PAGE gel image of the ethidium bromide stained and UV visualised 98 nt PCR fragment derived from pUC19 and the C5-amino-modified triphosphates 5a–8a. The full experimental conditions are given in Materials and Methods. Lane 1, molecular weight markers; lane 2, a PCR reaction containing all four natural triphosphates, dATP, dCTP, dGTP and dTTP; lane 3, a PCR reaction containing dATP, dCTP and dGTP, which does not result in formation of any product; lane 4, a PCR reaction containing dATP, dCTP, dGTP and 7a; lane 5, a PCR reaction containing dATP, dCTP, dGTP and 6a; lane 6, a PCR reaction containing dATP, dCTP, dGTP and 5a; lane 7, a PCR reaction containing dATP, dCTP, dGTP and 8a; lane 8, molecular weight markers. Triphosphates 5a and 6a (Fig. 2) are substrates for Taq polymerase during the PCR reaction but 7a and 8a are not.

Figure 4

PAGE gel image of the ethidium bromide stained and UV visualised 98 nt PCR fragment using pUC19 as template and the C5-imidazole-modified triphosphates 5b–8b, derived from imidazole 4-acetic acid. The full experimental conditions are given in Materials and Methods. Lane 1, molecular weight markers; lane 2, a PCR reaction containing all four natural triphosphates, dATP, dCTP, dGTP and dTTP; lane 3, a PCR reaction containing dATP, dCTP and dGTP, which does not result in formation of any product; lane 4, a PCR reaction containing dATP, dCTP, dGTP and 6b; lane 5, a PCR reaction containing dATP, dCTP, dGTP and 5b; lane 6, a PCR reaction containing dATP, dCTP, dGTP and 8b; lane 7, molecular weight markers. Triphosphates 5b and 6b (Fig. 2) are substrates for Taq polymerase during the PCR reaction but 8b is not.

Figure 5

PAGE gel image of the ethidium bromide stained and UV visualised 98 nt PCR fragment using pUC19 as template and the C5-imidazole-modified triphosphates 5c–8c, derived from urocanic acid. The full experimental conditions are given in Materials and Methods. Lane 1, molecular weight markers; lane 2, a PCR reaction containing all four natural triphosphates, dATP, dCTP, dGTP and dTTP; lane 3, a PCR reaction containing dATP, dCTP and dGTP, which does not result in formation of any product; lane 4, a PCR reaction containing dATP, dCTP, dGTP and 6c; lane 5, a PCR reaction containing dATP, dCTP, dGTP and 5c; lane 6, a PCR reaction containing dATP, dCTP, dGTP and 8c; lane 7, molecular weight markers. Triphosphates 5c and 6c (Fig. 2) are substrates for Taq polymerase during the PCR reaction but 8c is not.