Sanger Gap Sequencing for Genetic Alphabet Expansion of DNA

Abstract

Genetic alphabet expansion technology, creating new replicable and functional DNA molecules with unnatural base pairs (UBPs), is the novel promising research area of xenobiology. Recently, this technology has rapidly advanced, resulting in the need for a sequencing method for DNA molecules containing UBPs. However, all of the conventional sequencing methods, such as Sanger methods, are for four‐letter DNA molecules. Here, we present an improved Sanger sequencing method (Sanger gap sequencing) for DNAs containing our UBP, Ds‐Px, which appears as gaps in the sequencing peak patterns. By improving the sequencing reaction for efficient Ds‐Px pairing and using modified Px substrates, we have developed a sequencing method with increased processivity and clear gap patterns for multiple Ds‐Px pairs in various sequence contexts. This method is useful for UBP applications such as high‐affinity DNA aptamer generation and semisynthetic organism creation involving UBPs. In addition, through this research, we found that the side chains of UBs greatly affect the efficiency of UB pairings in replication, thus suggesting further development of UBPs.

Advancing Genetic Alphabet Expansion: Synthesis of 7‐(2‐Thienyl)‐Imidazo[4,5‐b]pyridine (Ds) and 4‐(4‐Pentyne‐1,2‐diol)‐1‐Propynyl‐2‐Nitropyrrole (Diol‐Px) for Use in Replicable Unnatural Base Pairs for PCR Applications

“…Among several modified dPxTP substrates (Kimoto et al., 2009; Okamoto et al., 2016; Yamashige et al., 2012), Diol‐dPxTP (Yamashige et al., 2012) shows the highest replication fidelity in PCR involving the Ds–Px pair and is widely used for PCR amplification of DNA containing Ds bases (Kimoto et al., 2012, 2013, 2020; Matsunaga et al., 2017, 2021). In developing the deoxyribonucleoside 2‐nitropyrrole (dPn; Hirao et al., 2007) as a pairing partner of Ds, dPn was found to be unstable to treatment with concentrated ammonia, especially at high temperatures.…”

“…In contrast, Px nucleotides are unstable under basic conditions, and thus DNA fragments with Px cannot be synthesized chemically (Hirao et al., 2007). To synthesize DNA fragments >100 nt that contain the Ds−Px pair, various methods can be employed (Kimoto et al., 2020; Kimoto et al., 2012). These involve polymerase reactions using dDsTP and dPxTP substrates with chemically synthesized Ds‐containing DNA templates (Fig.…”

“…Additionally, the 3′‐exonuclease activity of DNA polymerase plays a crucial role in removing both natural and unnatural misincorporated bases, thereby significantly improving replication fidelity. Replication fidelity is also influenced by modifications to the propynyl group in the Px base (Kimoto et al., 2020; Yamashige et al., 2012). When the combination of dDsTP and diol‐modified dPxTP (Diol‐dPxTP) is used, the selectivity of the Ds−Px pairing in PCR (conducted using DeepVent DNA polymerase, which possesses 3′‐exonuclease activity) exceeds 99.9% per replication cycle, and the misincorporation rate for these UB substrates opposite natural bases in templates is ∼0.01% per base pair per replication (Okamoto et al., 2016; Yamashige et al., 2012).…”

“…In (B) and (E), Ds‐containing DNAs are directly used for a template part, and thus additional dPxTP is not required. For (D), dPxTP is essential, since PCR‐amplified DNAs containing the Ds–Px pair are further used as templates for the intended full‐length DNAs (Kimoto et al., 2020; Kimoto et al., 2012).…”

From polymerase engineering to semi-synthetic life: artificial expansion of the central dogma

“…221,222 Hirao's group also developed a method for UBP sequencing, termed Sanger gap sequencing, in which the sequencing processivity was increased and modified Px analogs were used to generate clear gap patterns in the sequencing spectrum, which indicated the UBP positions. 223 Recently, Romesberg and coworkers reported the application of nanopore sequencing for the thorough analysis of DNA containing UBP NaM-TPT3. 25…”