Fluorescent base analogues

Contents

Introduction

Standard nucleobases (A, G, C, T and U) are not fluorescent under standard conditions. Fluorescent base analogues are structural analogues of the standard bases that are fluorescent, and form hydrogen bonds with complementary bases.

Fluorescent labels are commonly attached to the double helix at the end of a linker, which places the fluorophore relatively far from the DNA bases. This is useful in many circumstances, but sometimes it is necessary to incorporate a fluorophore closer to the DNA or RNA double helix, without perturbing the helix. As fluorescent base analogues are located rigidly in the double helix, their movement is restricted. This restricted movement results in a more predictable orientation of the fluorophore, an advantage in many applications such as fluorescence resonance energy transfer (FRET) and fluorescence anisotropy.

2-Aminopurine

2-Aminopurine (Figure 1) is the original, and most frequently used, fluorescent base analogue. It is highly fluorescent, and readily available.

Structure of 2-aminopurine (2AP), the first fluorescent base analogue, and structures of base pairs between 2-aminopurine and thymine (T) and cytosine (C)

Figure 1 | Structure of 2-aminopurine (2AP), the first fluorescent base analogue, and structures of base pairs between 2-aminopurine and thymine (T) and cytosine (C)

Like almost all fluorescent base analogues, 2-aminiopurine has a quantum yield that is heavily dependent on its environment. The DNA duplex quenches the fluorescence of more fluorescent base analogues, limiting their usefulness. This effect can be pronounced: 2-aminopurine has a good quantum yield of 0.68 in water, but this decreases to less than 0.02 in single-stranded DNA and less than 0.01 in double-stranded DNA.

Tricyclic cytosine analogues

The tricyclic cytosine analogues (Figure 2), intended for use in antisense therapy but recently discovered to be strongly fluorescent, are the only fluorescent base analogues to have quantum yields that are not affected appreciably by the environment. The quantum yield of the tricyclic cytosine analogue 1,3-diaza-2-oxophenothiazine (tC) does not vary much whether it is in monomeric form, in single- and double-stranded DNA; and whatever bases surround it.

Structures of cytosine (C), and the tricyclic cytosine analogues 1,3-diaza-2-oxophenothiazine (tC), 1,3-diaza-2-oxophenoxazine (tC<sup>O</sup>) and 7-nitro-1,3-diaza-2-oxophenothiazine (tC<sub>nitro</sub>) − the only fluorescent base analogues with quantum yields not appreciably affected by environment

Figure 2 | Structures of cytosine (C), and the tricyclic cytosine analogues 1,3-diaza-2-oxophenothiazine (tC), 1,3-diaza-2-oxophenoxazine (tCO) and 7-nitro-1,3-diaza-2-oxophenothiazine (tCnitro) − the only fluorescent base analogues with quantum yields not appreciably affected by environment

Like cytosine, the tricyclic cytosine analogues form hydrogen-bonding interactions with guanine (Figure 3), but not with adenine. If cytosine is replaced with tC in a DNA duplex, the DNA adopts the normal B-form, and only small distortions in the helix are observed around the base analogue: tC is therefore an excellent fluorescent base analogue.

Structure of the G·tC base pair

Figure 3 | Structure of the G·tC base pair

Relative to cytosine, tC has makes increased base stacking interactions in the DNA duplex, which means that duplexes containing tC are slightly more stable than analogous unmodified DNA.

The oxygen derivative of tC, tCO (Figure 2), is also an excellent base analogue and, with an extinction coefficient more than double that of tC (Table 1), tCO is the brightest fluorescent base analogue. The 7-nitro analogue of tC, tCnitro (Figure 2), is not fluorescent under standard conditions, but is used as a quencher, in combination with tC or tCO in a FRET pair.

Table 1Physical properties of fluorescent base analogues

Nameλmax / nm (absorption)λmax / nm (emission)E at λmaxΦ (solution)Φ (duplex)τ / ns (solution)τ / ns (duplex)
2-aminopyridine3033713 6000.68< 0.01--
tC39550540000.200.16–0.213.76.3
tCO36046590000.300.223.44.1
tCnitro440-5400----

The tricyclic cytosine analogues tC, tCO, and tCnitro can be incorporated into oligonucleotides during solid-phase synthesis via their commercially-available phosphoramidites (Figure 4).

Structures of tricyclic cytosine analogue phosphoramidites, for incorporation of the tricyclic cytosine analogues tC, tC<sup>O</sup> and tC<sub>nitro</sub> into oligonucleotides during phosphoramidite oligo synthesis

Figure 4 | Structures of tricyclic cytosine analogue phosphoramidites, for incorporation of the tricyclic cytosine analogues tC, tCO and tCnitro into oligonucleotides during phosphoramidite oligo synthesis

See also

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