3'-O-modified nucleotides as reversible terminators for pyrosequencing

Abstract

Pyrosequencing is a method used to sequence DNA by detecting the pyrophosphate (PPi) group that is generated when a nucleotide is incorporated into the growing DNA strand in polymerase reaction. However, this method has an inherent difficulty in accurately deciphering the homopolymeric regions of the DNA templates. We report here the development of a method to solve this problem by using nucleotide reversible terminators. These nucleotide analogues are modified with a reversible chemical moiety capping the 3'-OH group to temporarily terminate the polymerase reaction. In this way, only one nucleotide is incorporated into the growing DNA strand even in homopolymeric regions. After detection of the PPi for sequence determination, the 3'-OH of the primer extension products is regenerated through different deprotection methods. Using an allyl or a 2-nitrobenzyl group as the reversible moiety to cap the 3'-OH of the four nucleotides, we have synthesized two sets of 3'-O-modified nucleotides, 3'-O-allyl-dNTPs and 3'-O-(2-nitrobenzyl)-dNTPs as reversible terminators for pyrosequencing. The capping moiety on the 3'-OH of the DNA extension product is efficiently removed after PPi detection by either a chemical method or photolysis. To sequence DNA, templates containing homopolymeric regions are immobilized on Sepharose beads, and then extension-signal detection-deprotection cycles are conducted by using the nucleotide reversible terminators on the DNA beads to unambiguously decipher the sequence of DNA templates. Our results establish that this reversible-terminator-pyrosequencing approach can be potentially developed into a powerful methodology to accurately determine DNA sequences.

Fig. 1.

Structures of NRTs 3′-O-allyl-dNTP and 3′-O-(2-nitrobenzyl)-dNTP.

Fig. 2.

Synthesis of 3′-O-(2-nitrobenzyl)-dATP. (Step a) 2-nitrobenzyl bromide, tetrabutylammonium bromide, NaOH, in CH₂Cl₂ at room temperature for 1 h to produce compound 2 with a 95% yield. (Step b) Tetrabutylammonium fluoride in THF at room temperature for 1 h; methanolic ammonia and dioxane at 85–90°C for 12 h to produce compound 3 with a 56% yield. (Step c) POCl₃, PO(OMe)₃ at 0°C for 2 h; (Bu₃NH)₄P₂O₇, Bu₃N, triethylammonium bicarbonate, and NH₄OH at room temperature for 1.5 h to produce compound 4 with a 30% yield.

Fig. 3.

MALDI-TOF MS spectra of primer extension products with 3′-O-allyl-dNTPs (A–D) and 3′-O-(2-nitrobenzyl)-dNTPs (E–H). All eight 3′-O-modified nucleotides are quantitatively incorporated into the primers with high efficiency in the polymerase reaction, which indicates that the modified nucleotides are good substrates for the polymerase. The small peak near the 3′-O-(2-nitrobenzyl)-dNTP extension product corresponds to the photocleaved product generated during the laser desorption and ionization process used in MALDI-TOF MS.

Fig. 4.

The polymerase extension scheme using 3′-O-(2-nitrobenzyl)-dGTP (A Left–E Left) and MALDI-TOF MS spectra of the two consecutive extension products and their photocleavage products (A Right–E Right). (A) Primer for the polymerase extension reaction. (B) Primer extended with 3′-O-(2-nitrobenzyl)-dGTP to yield DNA extension product 2. (C) Product 2 photocleaved to yield photocleavage product 3. (D) Product 3 extended with another 3′-O-(2-nitrobenzyl)-dGTP to yield product 4. (E) Product 4 photocleaved to yield photocleavage product 5. After 30 s of irradiation with a laser at 355 nm, photocleavage is complete with all of the 3′-O-(2-nitrobenzyl)-group cleaved from the DNA extension products.

Fig. 5.

Signal intensity of luciferase catalyzed reactions using 0.5 nmol of dATP, 0.5 nmol of 3′-O-(2-nitrobenzyl)-dATP, 1.0 nmol of 3′-O-(2-nitrobenzyl)-dATP, 0.5 nmol of 3′-O-allyl-dATP, 1.5 nmol of 3′-O-allyl-dATP, and 1.5 nmol of ddATP. The results show that 3′-O-(2-nitrobenzyl)-dATP and 3′-O-allyl-dATP are not substrates of luciferase (NB, 2-nitrobenzyl).

Fig. 6.

Comparison of reversible terminator-pyrosequencing using 3′-O-allyl-dNTPs with conventional pyrosequencing using natural nucleotides. (A) The self-priming DNA template with stretches of homopolymeric regions (five A, two T, two G, and two C bases) was sequenced by using 3′-O-allyl-dNTPs. The homopolymeric regions are clearly identified, with each peak corresponding to the identity of each base in the DNA template. (B) Pyrosequencing data using natural nucleotides. The homopolymeric regions produced one large peak corresponding to the stretch of T bases and three smaller peaks for stretches of A, C, and G bases. However, it is very difficult to decipher the exact sequence from the data.

Fig. 7.

Comparison of reversible terminator-pyrosequencing using 3′-O-(2-nitrobenzyl)-dNTPs with conventional pyrosequencing using natural nucleotides (NB, 2-nitrobenzyl). (A) The self-priming DNA template with stretches of homopolymeric regions was sequenced by using 3′-O-(2-nitrobenzyl)-dNTPs. The homopolymeric regions are clearly identified, with each peak corresponding to the identity of each base in the DNA template. (B) Pyrosequencing data using natural nucleotides. The homopolymeric regions produced two large peaks corresponding to the stretches of G and A bases and five smaller peaks corresponding to stretches of T, G, C, A, and G bases. However, it is very difficult to decipher the exact sequence from the data.