Efficient enzymatic synthesis and dual-colour fluorescent labelling of DNA probes using long chain azido-dUTP and BCN dyes

Abstract

A sterically undemanding azide analogue of dTTP (AHP dUTP) with an alkyl chain and ethynyl attachment to the nucleobase was designed and incorporated into DNA by primer extension, reverse transcription and polymerase chain reaction (PCR). An azide-modified 523 bp PCR amplicon with all 335 thymidines replaced by AHP dU was shown to be a perfect copy of the template from which it was amplified. Replacement of thymidine with AHP dU increases duplex stability, accounting in part for the high incorporation efficiency of the azide-modified triphosphate. Single-stranded azide-labelled DNA was conveniently prepared from PCR products by λ-exonuclease digestion and streptavidin magnetic bead isolation. Efficient fluorescent labelling of single and double-stranded DNA was carried out using dyes functionalized with bicyclo[6.1.0]non-4-yne (BCN) via the strain-promoted alkyne-azide cycloaddition (SPAAC) reaction. This revealed that the degree of labelling must be carefully controlled to achieve optimum fluorescence and avoid fluorescence quenching. Dual-coloured probes were obtained in a single tube fluorescent labelling reaction; and varying the ratios of the two dyes provides a simple method to prepare DNA probes with unique fluorescent signatures. AHP dUTP is a versatile clickable nucleotide with potentially wide applications in biology and nanotechnology including single molecule studies and synthesis of modified aptamer libraries via SELEX.

Figure 1.

General scheme for fluorescent labelling of AHP-modified single and double-stranded DNA via the SPAAC reaction. The two curved arrows on the plasmid indicate the amplification direction and the grey section is the template region.

Figure 2.

(A) Chemical structure of azidomethyl (AM) dUTP. (B) Synthesis of azidohexanamidopropargyl deoxyuridine triphosphate (AHP dUTP).

Figure 3.

Primer extension using AHP dUTP (1.5 h). (A) Template T2 and primer P3. (B) Twenty percent denaturing PAGE analysis of reactions using Gotaq (Go, 72°C), Klenow (Kl, 37°C), KOD (KO, 72°C) and Therminator™ II (Th, 72°C) polymerases. Lane P: primer P3. (C) Reactions using Gotaq polymerase at 60°C. (D) Mass spectrum of AHP-modified fully extended product using Klenow (calculated mass: 10621).

Figure 4.

(A) Synthesis of BCN-functionalized fluorophores. (B) 5(6)-Carboxyfluorescein (FAM)-BCN (for ease of description 5(6)-FAM-BCN is written as FAM-BCN subsequently). (C) Cy5-BCN. (D) Cy3-BCN (19). (E) Rhodamine-B-BCN.

Figure 5.

Fluorescent labelling of AM and AHP-modified primer extension products. (A) Fully extended products from primer P3 and template T1. (B and C) Twenty percent denaturing PAGE analysis of samples before labelling and after labelled with Cy3-BCN at RT for 2 h. The unmodified product was used as a negative control.

Figure 6.

Reverse transcription using AHP dUTP. (A) RNA template T5 with primer P3. (B and C) Twenty percent denaturing PAGE analysis of reactions using AMV and M-MuLV (RNase H-) reverse transcriptases at 42°C for 15 h.

Figure 7.

PCR amplification using template T11, primers P9, P10 and KOD polymerase with different ratios of azide dUTPs to dTTP (10 nmol in total of azide dUTP/dTTP + 10 nmol each of dATP, dCTP and dGTP). (A) 1.5% agarose gel analysis. AM lanes: AM dUTP reactions; AHP lanes: AHP dUTP reactions. Lane L: 100 bp ladder. (B) Bar chart showing melting temperatures of AHP-modified amplicons. (C) Fluorescence melting curves of AHP-modified amplicons.

Figure 8.

Fluorescent labelling of PCR amplicons from template T11 containing different percentages of AHP dU. (A) Scheme for FAM-BCN labelling. (B) Fluorescence emission spectra of labelled PCR amplicons in TEAB buffer, excited at 480 nm. (C) Fluorescence intensity (relative fluorescence units, RFU) at 520 nm for each sample from B. Solvent: 75 mM TEAB buffer (pH 8.0).

Figure 9.

Dual labelling of 50% AHP-modified PCR amplicons from template T11 with different combinations of Cy3-BCN (100–0%) and FAM-BCN (0–100%). (A and B) 1.5% agarose gel analysis visualized on a transilluminator before staining and after staining with ethidium bromide. (C) Fluorescence spectra of labelled PCR amplicons in TEAB buffer (pH 8.0, samples were also analysed by agarose gel, shown in A and B), excited at 480 nm.

Figure 10.

λ-exonuclease digestion of PCR amplicons (81 bp, primer P6 and P7p) containing different percentages of AHP dU modifications and re-annealing of the modified single strands to complementary template T8. 2% agarose gel analysis containing SYBR Gold stain. PCR lanes: PCR amplicons; single strand lanes: PCR amplicons after exonuclease digestion; re-annealing lanes, ssDNA re-annealed with T8. Lane L: 25 bp ladder. D = duplex products; T + P = excess primer annealed to template T8; T = template T8.

Figure 11.

Fluorescent labelling of ssDNA (81-mer) containing different percentages of AHP dU. (A and B) 12% native PAGE analysis visualized on a transilluminator, before staining and after staining with SYBR Gold. Single strand lanes: single-stranded products from exonuclease digestion; FAM-BCN labelled lanes: ssDNA labelled with FAM-BCN at 55°C for 1 h. Lane L: 25 bp ladder.

Figure 12.

Dual labelling of 50% AHP-modified single strands from streptavidin magnetic bead separation with different combinations of Cy5-BCN (100% to 0%) and Cy3-BCN (0% to 100%). (A) Scheme for dual labelling and re-annealing. (B) Relative fluorescence intensity for the re-annealed duplex bands (DB2) quantified by gel analysis in Cy5 and Cy3 channels (for native PAGE analysis see Supplementary Figure S17, ESI).