Labeled nucleoside triphosphates with reversibly terminating aminoalkoxyl groups

Abstract

Nucleoside triphosphates having a 3'-ONH₂ blocking group have been prepared with and without fluorescent tags on their nucleobases. DNA polymerases were identified that accepted these, adding a single nucleotide to the 3'-end of a primer in a template-directed extension reaction that then stops. Nitrite chemistry was developed to cleave the 3'-ONH₂ group under mild conditions to allow continued primer extension. Extension-cleavage-extension cycles in solution were demonstrated with untagged nucleotides and mixtures of tagged and untagged nucleotides. Multiple extension-cleavage-extension cycles were demonstrated on an Intelligent Bio-Systems Sequencer, showing the potential of the 3'-ONH₂ blocking group in "next generation sequencing."

Figure 1

Schematic showing sequencing using cyclic reversible termination in the general case, together with the structure of triphosphates having a 3′-ONH₂ blocking group.

Figure 2

Reversible terminators discussed in this manuscript. 1-4 untagged, 5-8 tagged. Fluorophores: 5a) Cy3; 5b) 5-FAM; 5c) 6-FAM; 6) Cy3.5; 7) Cy5; 8) BODIPY-FL C₅.

Figure 3

Cycling with untagged 3′-ONH₂ triphosphates on two-dimensional array on the PinPoint Sequencer (Intelligent Bio-Systems). A. Self priming templates (30–33) used in sequencing experiments. Black underlined sequence indicates the annealed primer, the gray colored bases indicate 7 sequencing cycles in which a mixture of all four untagged 3′-ONH2 triphosphates (1-4) were used. Color indicates readout positions (cycle 8) for which a mixture of labeled ddNTPs was used. B. Bar graphs showing 4 – channel fluorescence intensity observed for templates 30 – 33. The calls (the channel with the highest signal) correctly detected the nucleotide present at position 8 for all four templates and indicate that the preceding 7 cycles of incorporation and cleavage of the correct 3′-ONH₂ were complete and in phase. The residual signals observed in other channels is most likely due to a combination of incomplete background/crosstalk correction and/or some dephasing. For view of instrument and more experimental details, see text and Fig. E16 and E17 (Supplemental).

Figure 4

Reversible terminator sequencing cycle using 7-deaza-dATP-ONH₂-Cy5 (7), dCTP-ONH₂-Cy3.5 (6), dATP-ONH₂ (3), dCTP-ONH₂ (2) and Therminator (0.5 U/reaction). The cycles involve (a) adding reversibly terminated triphosphate to a primer annealed to a template attached via a biotin tag to a streptavidin bead (to give an n+1 band that migrates more slowly if the triphosphate is tagged); (b) treating the beads with buffered sodium nitrite (the active reagent is HONO) to generate a free extendable 3′-OH end; (c) cleaving the 1,2-diol side chain with aqueous sodium periodate (50 mM) to release the fluorophore; (d) resuming extension. The tagged and untagged triphosphates were alternated. If a tagged triphosphate was added first, the untagged triphosphate was added second. If an untagged triphosphate was added first, the tagged triphosphate was added second. For more experimental details, see Fig. E15 (Supplemental).

Scheme 1

Synthetic strategy for managing four reactive units in a taggable nucleoside triphosphate (-ONH₂, triphosphate, 1,2-diol cleavable linker, and fluorophore) illustrated for the thymidine analog. Analogous syntheses were done with 5-iodo-2′-deoxycytidine, 7-deaza-7-iodo-2′-deoxyadenosine, and 7-deaza-7-iodo-2′-deoxyguanosine (see Supplemental). Conditions: see Supplemental.

Scheme 2

Synthesis of the untagged 2′-deoxycytidine triphosphate having just two reactive units (-ONH₂ and triphosphate). Analogous syntheses were done with the other three natural nucleosides (see Supplemental). Conditions: (a) (1) BzOH, Ph₃P, DIAD, THF, 0°C to RT, 16 hours; (2) NaOMe, MeOH, RT, 2 hours (62% overall); (b) (1) N-hydroxyphthalimide, Ph₃P, DIAD, THF, 0°C to RT, 16 hours; (2) HCl, MeOH, RT, 10 minutes (63% overall); (c) (1) MeNH₂, H₂O, RT, 10 minutes; (2) acetone, H₂O, RT, 3 hours (71% overall after HPLC); (d) (1) 2-chloro-4H-1,3,2-benzodioxaphosphorin-4-one, pyridine/dioxane, RT, 15 minutes; (2) (Bu₃N)₄P₂O₇, DMF, RT, 20 minutes; (3) iodine, pyridine/H₂O, 20 minutes (38% overall after double HPLC).