Base recognition by L-nucleotides in heterochiral DNA

Abstract

Thermal stabilities of homo- and heterochiral DNA duplexes with and without a base pair mismatch at the chiral modification site were evaluated. The results indicated that the L-nucleotide residue in heterochiral duplexes retains its selectivity toward the complementary D-nucleotide residue.

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An L-nucleotide consisting of L-deoxyribose as the chiral unit is the enantiomer of natural D-nucleotide. The nucleic acids which construct living bodies are only composed of D-nucleotides. Therefore, the D-homochirality is essential for the formation of higher-order structures and the biological functions of nucleic acid. L-Homochiral DNA forms left-handed duplexes with complementary L-homochiral sequences,¹ whereas they cannot hybridize with complementary D-homochiral sequences.² The unnatural L-DNA is not recognized by natural enzymes consisting of L-amino acids and thus show excellent resistance to enzymatic degradation. By using such characteristics, L-DNA has been utilized for the development of various tools, such as anti-HIV (human immunodeficiency virus) agents,³aptamers,⁴ molecular beacons,⁵ and microarrays.⁶

The effect of the substitution of D-nucleotides with their L-counterparts on the duplex stability has been also investigated. Heterochiral DNAs that contain an L-nucleotide within natural D-sequences were found to retain the duplex structure, in which the L-nucleotide residue forms a stable Watson–Crick type base pair with its natural complementary residue.⁷ These results suggest that the L-nucleotide residue in heterochiral DNA may possess base-discriminating ability, although the duplex stability is somewhat decreased compared to that of parental D-homochiral DNA.⁸ Similar results showing that chiral modification decreases the thermal stability of duplexes were also reported by other groups.⁹ However, those reports simply described the duplex stability of heterochiral oligodeoxynucleotides (ODNs) containing L-nucleotides at a specific site, and the base-discriminating ability of L-nucleotides in heterochiral DNA has not been reported.

Here we report the duplex stability of heterochiral DNA, each of which has an L-nucleotide at a different site, and the base pairing selectivity of the L-nucleotides in the heterochiral DNA.

We synthesized homochiral and some heterochiral template strands (Table 1) and investigated their duplex forming ability with the complementary strands by conducting UV-melting experiments (Fig. 1). Each heterochiral duplex showed a cooperative transition as well as a D-homochiral duplex. Table 1 summarizes the effects of the substitution of D-nucleotide with L-nucleotide at different sites of the D-homochiral template strand on the duplex stability. The substitution of D-nucleotide with L-nucleotide resulted in a decrease of T_m values in the range of 3.9 to 9.5 °C. It was clearly shown that the heterochiral duplexes have lower T_m values than the parental ones, and the decrease of T_m values shows the sequence dependence as reported previously.⁸

Fig. 1 UV-melting profiles of homo- (duplex 1) and heterochiral duplexes (duplexes 2–5). Samples contained 6 μM duplex in 10 mM MgCl₂, 100 mM NaCl, and 70 mM MOPS (pH 7.1).

Table 1 UV-melting points of homo- and heterochiral duplexes^a

a Samples contained 6 μM duplex in 10 mM MgCl2, 100 mM NaCl, and 70 mM MOPS (pH 7.1). b Melting temperature difference from the homochiral duplex.
Duplex	Template strand	Complementary strand	T m (°C)	ΔTm (°C)^b
Homochiral strand
1	d(GCGTCTAAA)	d(TTTAGACGC)	42.1	—
Heterochiral strand
2	d(GCGTCTLAAA)	d(TTTAGACGC)	33.6	−8.5
3	d(GCLGTCTAAA)		32.6	−9.5
4	d(GCGTLCTAAA)		38.2	−3.9
5	d(GCGTCLTAAA)		33.9	−8.2

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To investigate the base pairing selectivity of L-nucleotides in heterochiral duplexes, we examined the thermal stability of the homo- and heterochiral duplexes containing a base pair mismatch at the chiral modification site. In the case of homochiral duplexes (Fig. 2a–d), the T_m values were considerably decreased by the introduction of a base pair mismatch (ΔT_m>10 °C), which means that D-nucleotides in D-homochiral strands have high base-discriminating ability (Fig. 2a–d). Notably, the decrease of the T_m value by substituting D-nucleotide with L-nucleotide is smaller (Table 1) than that by introducing a base pair mismatch (Table S1†). This result suggests that L-nucleotide residues in heterochiral duplexes form hydrogen bonds with their complementary bases. All of the fully matched heterochiral duplexes gave the highest T_m values among the four duplexes (Fig. 2e–h; yellow bars) in each series, similarly to the fully matched homochiral ones. Particularly, the fully matched heterochiral duplex containing L-dC (Fig. 2f; yellow bars) showed the similar base-discriminating ability to the fully matched homochiral one (Fig. 2b; yellow bars). Furthermore, the fully matched heterochiral duplexes containing L-dA and L-T also showed higher T_m values than the corresponding mismatched duplexes (Fig. 2e and h; yellow bars), although their respective selectivities to the complementary T and dA are somewhat lower than those of the corresponding homochiral duplexes. (Fig. 2a and d). In the case of chiral modification of dG, L-dG was unable to discriminate between dC and dG. These results suggest that L-nucleotides in heterochiral duplexes generally retain their base-discriminating ability, which is probably based on Watson–Crick type hydrogen bonding in a manner similar to D-nucleotides in D-homochiral duplexes, although the base pairing selectivity may be affected by the kind of L-nucleotide and its neighboring bases. Heterochiral duplexes containing L-dA, L-dG, and L-T (Fig. 2e, g, and h, respectively) have lower base pairing selectivity than their corresponding homochiral duplexes. On the other hand, the L-dC residue in the heterochiral strand (Fig. 2f) shows similar base-discriminating ability to the D-dC residue in the corresponding homochiral strand (Fig. 2b), and their ΔT_m values by base pair mismatch are similar (Table S1†). Considering the quite small ΔT_m value of duplex 4 (Table 1), the L-nucleotide in a heterochiral duplex may have the strong base-discriminating ability when the chiral modification does not cause marked duplex destabilization.

Fig. 2 Effects of base pair mismatch of D- (a–d) and L-nucleotide (e–h) on duplex stability. Samples contained 6 μM duplex in 10 mM MgCl₂, 100 mM NaCl, and 70 mM MOPS (pH 7.1). Yellow bars denote T_m values of fully matched duplexes, and blue bars denote T_m values of mismatched duplexes.

In conclusion, we found that heterochiral duplexes have generally lower stability than parental homochiral duplexes and L-nucleotides in heterochiral ODNs possess the base pairing selectivity although the selectivity depends on the kind of L-nucleotide and its neighboring bases. The base-discriminating ability of L-nucleotide in heterochiral ODNs may shed light on their potential use as a tool for molecular recognition.

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Footnote

† Electronic supplementary information (ESI) available: MALDI-TOF mass data of oligodeoxynucleotides and melting points of the duplexes. See DOI: 10.1039/c2ra01013e

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