Faithful PCR Amplification of an Unnatural Base‐Pair Analogue with Four Hydrogen Bonds

Abstract

In vitro replication of an unnatural imidazopyridopyridine:naphthyridine base pair, (i.e., ImN(N):NaO(O)), having four hydrogen bonds was investigated. Kinetic studies of single-nucleotide insertion revealed that ImN(N) and NaO(O) were recognized as complementary bases by an exonuclease-deficient Klenow fragment with higher specificity and efficiency than two previously described pairs (ImN(O):NaO(N) and ImO(N):NaN(O)) because of higher thermal and thermodynamic stabilities and the DAAD:ADDA (D=donor, A=acceptor) hydrogen-bonding pattern of the ImN(N):NaO(O) pair. Faithful polymerase chain reaction (PCR) amplification of a DNA fragment containing the ImN(N):NaO(O) pair was achieved by using DNA polymerases possessing 3'→5' exonuclease activity (≈99.5 % per doubling).

Unnatural Base Pairs for Synthetic Biology

“…When a mixture of 2 prepared from 2′-deoxyinosine and 3 prepared from 2,4-dichloropyrimidine is 37) The resulting 1 is then converted into the corresponding phosphoramidite unit and 5′-triphosphate under the appropriate conditions. 44,45) For the synthesis of NaO O nucleoside 6, which is an unusual C-nucleoside, the palladium-catalyzed Heck reaction was envisioned. As illustrated in Chart 2, 3-iodo-1,8-naphthyridine derivative 7 prepared from 2-amino-7-hydroxy-1,8-naphthyridine and glycal 8 are prepared.…”

“…In a similar manner as for 1, 6 is converted into the corresponding phosphoramidite unit and 5′-triphosphate for enzymatic evaluation. 39,45) …”

“…N : NaO O Pair To investigate the efficiency and selectivity of the newly designed ImN N : NaO O pair in in vitro replication systems, we examined single nucleotide insertion using KF polymerase, and the kinetic parameters, such as the Michaelis constant (K m ), the maximum rate of the enzyme reaction (V max ), and the incorporation efficiency (V max /K m ), for the ImN N : NaO O pair were determined and compared with those of the two previous Im : Na pairs 45) (Fig. 6).…”

Investigation of the stability of D5SIC-DNAM-incorporated DNA duplex in Taq polymerase binary system: a systematic classical MD approach

“…2–5 In biological process, DNA polymerases are specific towards four nucleotides (A, T, G, C) and two base-pairings (A–T and G–C). 1–6 The concept of incorporating synthetic molecules as base pairs, termed Unnatural Base-Pairs (UBPs), 7–42 depends upon a crucial prerequisite: these synthetic entities must undergo a thorough selection process orchestrated by polymerase enzymes. 43–50 In essence, the viability of these synthetic molecules as base pairs is contingent upon the polymerase's ability to discriminate and incorporate them into the growing DNA strand with a high degree of accuracy and efficiency.…”

Investigation of dynamical flexibility of D5SIC-DNAM inside DNA duplex in aqueous solution: a systematic classical MD approach

“…3–5 As a result, scientists have dedicated considerable effort over the last decade to discovering fifth and sixth nucleotides that can form a third, unnatural base pair (UBP) with increased functionality that can be orthogonally replicated in DNA. 4,6–37 This would also expand the usefulness of nucleic acids for biological and biotechnological applications.…”