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.

Expanding the genetic alphabet enhances DNA recombinant technologies by introducing unnatural base pairs (UBPs) beyond the standard A-T and G-C pairs, leading to biomaterials with novel and increased functionalities. Recent developments include UBPs that effectively function as a third base pair in replication, transcription, and/or translation processes. One such UBP, Ds-Px, demonstrates extremely high specificity in replication. Chemically synthesized DNA fragments containing Ds bases are amplified by PCR with the 5'-triphosphates of Ds and Px deoxyribonucleosides (dDsTP and dPxTP). The Ds-Px pair system has applications in enhanced DNA data storage, generation of high-affinity DNA aptamers, and incorporation of functional elements into RNA through transcription. This protocol describes the synthesis of the amidite derivative of Ds (dDs amidite), the triphosphate dDsTP, and the diol-modified dPxTP (Diol-dPxTP) for PCR amplifications involving the Ds-Px pair. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Synthesis of Ds deoxyribonucleoside (dDs) Basic Protocol 2: Synthesis of dDs amidite Basic Protocol 3: Synthesis of dDs triphosphate (dDsTP) Basic Protocol 4: Synthesis of Pn deoxyribonucleoside (4-iodo-dPn) Basic Protocol 5: Synthesis of acetyl-protected diol-modified Px deoxyribonucleoside (Diol-dPx) Basic Protocol 6: Synthesis of Diol-dPx triphosphate (Diol-dPxTP) Basic Protocol 7: Purification of triphosphates Support Protocol 1: Synthesis of Hoffer's chlorosugar Support Protocol 2: Preparation of 0.5 M pyrophosphate in DMF Support Protocol 3: Preparation of 2 M TEAB buffer.

Strict Interactions of Fifth Letters, Hydrophobic Unnatural Bases, in XenoAptamers with Target Proteins.

XenoAptamers are DNA fragments containing additional letters (unnatural bases, UBs) that bind specifically to their target proteins with high affinities (sub-nanomolar KD values). One of the UBs is the highly hydrophobic 7-(2-thienyl)imidazo[4,5-b]pyridine (Ds), which significantly increases XenoAptamers' affinities to targets. Originally, Ds was developed as a third base pair with a complementary UB, 2-nitro-4-propynylpyrrole (Px), for replication, and thus it can be used for aptamer generation by an evolutional engineering method involving PCR amplification. However, it is unclear whether the Ds base is the best component as the hydrophobic fifth-letter ligand for interactions with target proteins. To optimize the ligand structure of the fifth letter, we prepared 13 Ds variants and examined the affinities of XenoAptamers containing these variants to target proteins. The results obtained using four XenoAptamers prepared by the replacement of Ds bases with variants indicated that subtle changes in the chemical structure of Ds significantly affect the XenoAptamer affinities. Among the variants, placing either 4-(2-thienyl)pyrrolo[2,3-b]pyridine (Ys) or 4-(2-thienyl)benzimidazole (Bs) at specific Ds positions in each original XenoAptamer greatly improved their affinities to targets. The Ys and Bs bases are variants derived by replacing only one nitrogen with a carbon in the Ds base. These results demonstrate the strict intramolecular interactions, which are not simple hydrophobic contacts between UBs and targets, thus providing a method to mature XenoAptamers' affinities to targets.

Success probability of high-affinity DNA aptamer generation by genetic alphabet expansion.

Nucleic acid aptamers as antibody alternatives bind specifically to target molecules. These aptamers are generated by isolating candidates from libraries with random sequence fragments, through an evolutionary engineering system. We recently reported a high-affinity DNA aptamer generation method that introduces unnatural bases (UBs) as a fifth letter into the library, by genetic alphabet expansion. By incorporating hydrophobic UBs, the affinities of DNA aptamers to target proteins are increased over 100-fold, as compared with those of conventional aptamers with only the natural four letters. However, there is still plenty of room for improvement of the methods for routinely generating high-affinity UB-containing DNA (UB-DNA) aptamers. The success probabilities of the high-affinity aptamer generation depend on the existence of the aptamer candidate sequences in the initial library. We estimated the success probabilities by analysing several UB-DNA aptamers that we generated, as examples. In addition, we investigated the possible improvement of conventional aptamer affinities by introducing one UB at specific positions. Our data revealed that UB-DNA aptamers adopt specific tertiary structures, in which many bases including UBs interact with target proteins for high affinity, suggesting the importance of the UB-DNA library design. This article is part of the theme issue 'Reactivity and mechanism in chemical and synthetic biology'.

High-affinity five/six-letter DNA aptamers with superior specificity enabling the detection of dengue NS1 protein variants beyond the serotype identification.

Genetic alphabet expansion of DNA by introducing unnatural bases (UBs), as a fifth letter, dramatically augments the affinities of DNA aptamers that bind to target proteins. To determine whether UB-containing DNA (UB-DNA) aptamers obtained by affinity selection could spontaneously achieve high specificity, we have generated a series of UB-DNA aptamers (KD: 27-182 pM) targeting each of four dengue non-structural protein 1 (DEN-NS1) serotypes. The specificity of each aptamer is remarkably high, and the aptamers can recognize the subtle variants of DEN-NS1 with at least 96.9% amino acid sequence identity, beyond the capability of serotype identification (69-80% sequence identities). Our UB-DNA aptamers specifically identified two major variants of dengue serotype 1 with 10-amino acid differences in the DEN-NS1 protein (352 aa) in Singaporeans' clinical samples. These results suggest that the high-affinity UB-DNA aptamers generated by affinity selection also acquire high target specificity. Intriguingly, one of the aptamers contained two different UBs as fifth and sixth letters, which are essential for the tight binding to the target. These two types of unnatural bases with distinct physicochemical properties profoundly expand the potential of DNA aptamers. Detection methods incorporating the UB-DNA aptamers will facilitate precise diagnoses of viral infections and other diseases.

Cognate base-pair selectivity of hydrophobic unnatural bases in DNA ligation by T4 DNA ligase.

We present cognate base pair selectivity in template-dependent ligation by T4 DNA ligase using a hydrophobic unnatural base pair (UBP), Ds-Pa. T4 DNA ligase efficiently recognizes the Ds-Pa pairing at the conjugation position, and Ds excludes the noncognate pairings with the natural bases. Our results indicate that the hydrophobic base pairing is allowed in enzymatic ligation with higher cognate base-pair selectivity, relative to the hydrogen-bond interactions between pairing bases. The efficient ligation using Ds-Pa can be employed in recombinant DNA technology using genetic alphabet expansion, toward the creation of semi-synthetic organisms containing UBPs.

Inhibition of dengue virus RNA synthesis by an adenosine nucleoside.

We recently reported that (2R,3R,4R,5R)-2-(4-amino-pyrrolo[2,3-d]pyrimidin-7-yl)-3-ethynyl-5-hydroxy-methyl-tetrahydro-furan-3,4-diol is a potent inhibitor of dengue virus (DENV), with 50% effective concentration (EC(50)) and cytotoxic concentration (CC(50)) values of 0.7 microM and >100 microM, respectively. Here we describe the synthesis, structure-activity relationship, and antiviral characterization of the inhibitor. In an AG129 mouse model, a single-dose treatment of DENV-infected mice with the compound suppressed peak viremia and completely prevented death. Mode-of-action analysis using a DENV replicon indicated that the compound blocks viral RNA synthesis. Recombinant adenosine kinase could convert the compound to a monophosphate form. Suppression of host adenosine kinase, using a specific inhibitor (iodotubercidin) or small interfering RNA (siRNA), abolished or reduced the compound's antiviral activity in cell culture. Studies of rats showed that (14)C-labeled compound was converted to mono-, di-, and triphosphate metabolites in vivo. Collectively, the results suggest that this adenosine inhibitor is phosphorylated to an active (triphosphate) form which functions as a chain terminator for viral RNA synthesis.

An adenosine nucleoside inhibitor of dengue virus.

Dengue virus (DENV), a mosquito-borne flavivirus, is a major public health threat. The virus poses risk to 2.5 billion people worldwide and causes 50 to 100 million human infections each year. Neither a vaccine nor an antiviral therapy is currently available for prevention and treatment of DENV infection. Here, we report a previously undescribed adenosine analog, NITD008, that potently inhibits DENV both in vitro and in vivo. In addition to the 4 serotypes of DENV, NITD008 inhibits other flaviviruses, including West Nile virus, yellow fever virus, and Powassan virus. The compound also suppresses hepatitis C virus, but it does not inhibit nonflaviviruses, such as Western equine encephalitis virus and vesicular stomatitis virus. A triphosphate form of NITD008 directly inhibits the RNA-dependent RNA polymerase activity of DENV, indicating that the compound functions as a chain terminator during viral RNA synthesis. NITD008 has good in vivo pharmacokinetic properties and is biologically available through oral administration. Treatment of DENV-infected mice with NITD008 suppressed peak viremia, reduced cytokine elevation, and completely prevented the infected mice from death. No observed adverse effect level (NOAEL) was achieved when rats were orally dosed with NITD008 at 50 mg/kg daily for 1 week. However, NOAEL could not be accomplished when rats and dogs were dosed daily for 2 weeks. Nevertheless, our results have proved the concept that a nucleoside inhibitor could be developed for potential treatment of flavivirus infections.