Enine tilt the bases relative to each other yielding a base pair that effectively contains only a single hydrogen bond. However, 2-thio-T base pairs perfectly well with A, as shown in Figure 1. SBC 20mers annealed against a DNA 20mer target exhibited Tm values 10higher than the corresponding DNA-DNA hybrid, whereas the SBC-SBC hybrid yielded Tm values up to 30lower.1 2-Thio-dT (3) is best deprotected under mild conditions using ammonium hydroxide at room temperature. Pac-2-amino-dA (2) is also efficiently deprotected in ammonium hydroxide and so is compatible with 2-thio7
where A is 2-Amino-dA. The resulting melting curves shown in Figure 4 and summarized above indicate that the increase in melting temperature inherent in the 2-Amino-A-T base pair is indeed cumulative. An increase in Tm of 3.0was observed with three substitutions fairly evenly spaced throughout our test oligo to give an average of 1.0per insertion relative to the control. When a further two substitutions were made less optimally at adjacent sites in our test oligo, the increase in Tm was 5.1relative to the control, with the average still at 1.1372540-25-4 site 0per insertion. Our simple experiment demonstrates that the increase in melting temperature
dT. But is this combination of Pac-2-aminodA and 2-thio-dT capable of producing SBC oligos The following experiment with SBC oligos was carried out. The sequences were:
SBC fwd: 5′-TCT GAA GCT GTT ACT CCG-3′ SBC rev: 5′-CGG AGT AAC AGC TTC AGA-3′ Reg AT fwd: 5′-TCT GAA GCT GTT ACT CCG-3′ Reg AT rev: 5′-CGG AGT AAC AGC TTC AGA-3′
FIGURE 5: MELTING CURVES USING SBC OLIGOS AND CONTROLS
– SBC fwd/SBC rev – Reg AT bases fwd/rev – SBC fwd/AT bases rev – AT bases fwd/SBC rev
where A and T are 2-Amino-dA and 2-Thio-dT, respectively. The oligos were deprotected in ammonium hydroxide at room temperature for 24 hours and purified on Glen-PakTM cartridges. Figure 5 shows the melting plot which has been smoothed and normalized to 0.386 AU at 260 nm at 20 . The color code is:
Blue: SBC fwd/SBC rev Teal: Reg AT bases fwd/rev Green: SBC fwd/AT bases rev Red: AT bases fwd/SBC rev Tm 46.0 59.0 64.0 64.5
The runs were performed in degassed 0.1 M Tris-HCl, pH 7.4 with the oligos at 1.25 concentration using a Jasco V-530 fitted with a Peltier temperature control unit.
In our experiment, the SBC-SBC hybrid yielded a Tm 13lower than the control standard oligo hybrid, while the SBC-regular hybrids had Tm 5higher than the control oligo. This experiment confirms that our Pac-2-amino-dA monomer allows relatively straightforward production of SBC oligos. CONCLUSION We are happy to offer an alternative monomer for the synthesis of oligos containing 2-Amino-dA.446022-33-9 Biological Activity Our goals of designing a monomer that is fully compatible with regular oligo synthesis and deprotection have been achieved, as evidenced by the simple preparation of oligos with multiple 2-Amino-dA residues, as well as the preparation of SBC oligos.PMID:30649797 We thank Stefan Pitsch of Pitsch Nucleic Acids for his help in the design and production of Pac-2-Amino-dA-CE Phosphoramidite. Reference:
1. I.V. Kutyavin, et al., Biochemistry, 1996, 35, 11170-11176.
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