Rewriting the Central Dogma with Synthetic Genetic Polymers

Abstract

DNA and RNA are ubiquitous molecules responsible for storage and transmission of genetic information and together comprise the central dogma of molecular biology. However, the recent emergence of synthetic genetic polymers is providing an opportunity to challenge the fundamental principles of life. Herein, we describe the ongoing attempts to rewrite the central dogma with 4′-thioDNA and 4′-thioRNA, which feature a sulfur instead of an oxygen atom in the furanose ring moiety. Using reconstituted Escherichia coli gene expression machinery, studies have shown that the genetic information conserved in 4′-thioDNA can be transcribed to 4′-thioRNA and eventually translated into protein, mirroring the processes that occur in nature. Such studies underscore the feasibility of controlling life by substances other than DNA and RNA.

1. Introduction

The dramatic progress of life science research witnessed in the latter half of the 20th century has led to the identification of a range of attractive novel biological targets, whose modulation may enable novel therapeutic options for many diseases. To address various drug targets, researchers have been intensely investigating so-called “new therapeutic modalities” as well as drug delivery systems in recent years.¹⁾ In this review, we focus on research conducted on synthetic genetic polymers that might replace natural DNA and RNA—the ubiquitous molecules that store and transmit genetic information—as candidates for new therapeutic modalities.

The genetic information stored in DNA is transferred to RNA, and eventually translated into proteins. This flow, known as the central dogma of molecular biology, is a fundamental principle that forms the basis of life.²⁾ However, the recent emergence of synthetic genetic polymers that have DNA- and RNA-like functions may provide access to alternative gene expression systems. Therefore, intense efforts have focused on identifying synthetic polymers that may function in the sequential flow of genetic information based on the central dogma.³⁾

Meanwhile, our group has successfully proposed 4′-thioDNA and 4′-thioRNA, which feature a sulfur instead of an oxygen atom in the furanose ring moiety, as bioisosteres of natural DNA/RNA^4–20) (Fig. 1). The 4′-thioDNA/4′-thioRNA analogues exhibit high duplex forming ability, as well as stability against nuclease-mediated hydrolysis.^4,8,11–13) By taking advantage of such favorable properties, we have successfully applied 4′-thioDNA and 4′-thioRNA in the field of drug discovery.^{4–7,9–11,14–18,20)} Furthermore, we recently succeeded in transcribing the genetic information stored in 4′-thioDNA into 4′-thioRNA, and translating it into protein by using 2′-deoxy-4′-thionucleoside 5′-triphosphates (d^SNTPs)^14,15) and 4′-thioribonucleoside 5′-triphosphates (r^SNTPs)^5,10) as building blocks of 4′-thioDNA and 4′-thioRNA, respectively¹⁹⁾ (Figs. 1b, c). Herein, we describe how the central dogma is being rewritten with synthetic genetic polymers such as 4′-thioDNA/4′-thioRNA.

Fig. 1. 4′-ThioDNA Provides Genetic Information to 4′-ThioRNA for Protein Synthesis

(a, b) Chemical structures of 4′-thioDNA and 4′-thioRNA (a), and 2′-deoxy-4′-thionucleoside 5′-triphosphates (d^SNTPs) and 4′-thioribonucleoside 5′-triphosphates (r^SNTPs) (b). (c) Rewriting the central dogma with 4′-thioDNA and 4′-thioRNA. Base = A, G, C, T, or U.

2. Rewriting the Central Dogma with Synthetic Genetic Polymers

Before discussing in detail our studies on 4′-thioDNA and 4′-thioRNA, we wish to briefly summarize some of the pioneering research in this field.

2.1. Nucleobase-Modified Genetic Polymers

The most important breakthroughs towards rewriting the central dogma were achieved using nucleobase-modified analogues. The Romesberg group reported the creation of a semi-synthetic organism that could store and retrieve expanded genetic information based on a non-hydrogen-bonded unnatural base pair indicated as NaM : TPT3^21,22) (Fig. 2a). They successfully used an exogenously expressed nucleoside triphosphate transporter from Phaeodactylum tricornutum (PtNTT2) to import deoxyribo- and ribotriphosphates of various nucleobase modified nucleosides (dNaMTP, dTPT3TP, rNaMTP, and rTPT3TP) into Escherichia coli (E. coli), where the endogenous replication, translation, and transcription machinery used them to encode and decode an expanded unnatural genetic codon. These studies have had an enormous impact on the field of synthetic biology, although some researchers consider the so-created organism to be “alien.”²³⁾

Fig. 2. Structures of Nucleobase-Modified Elements as Components of Synthetic Genetic Polymer Alternatives to Natural DNA/RNAs

a) Left, NaM : TPT3 pair; right, a semi-synthetic organism with NaM : TPT3. b) isoG : isoC pair. c) s : y pair. d) 5-Substituted uracil bases. e) 5-Substituted cytosine bases. f) 7-Deazaadenine. g) 7-Deazaguanine. h) Size-expanded Watson–Crick like base pair.

Other types of nucleobase-modified elements have been implemented in a separate but coupled transcription–translation system, leading to the incorporation of a non-canonical amino acid into a protein in vitro. For example, an isoG : isoC pair (Fig. 2b), which has unnatural hydrogen-bonding geometry, has been recognized as a complementary base pair by polymerases in both in vitro replication and in vitro transcription systems.²⁴⁾ In addition, the unnatural 3-iodotyrosine has been incorporated into a peptide fragment in an in vitro translation system by using a combination of a 54-mer mRNA with an isoC and tRNA with an isoGUC anticodon.²⁵⁾ A non-hydrogen-bonded s : y pair designed by Hirao et al. has also been shown to function in an expanded genetic code in an E. coli cell-free translation system²⁶⁾ (Fig. 2c).

Although the corporation of these synthetic elements is limited by the hierarchy of replication, the E. coli gene expression machinery has been proven to accept the replacement of one of the four natural nucleobases with 5-substituted pyrimidine analogues, such as 5-chloro- and 5-hydroxymethyluracil (Fig. 2d), and 5-hydroxymethylcytosine (Fig. 2e), throughout the bacterial entire genome.^27–29) In addition, all four nucleobases can be substituted with 5-chlorouracil, 5-fluorocytosine (Fig. 2e), 7-deazaadenine (Fig. 2f), and 7-deazaguanine (Fig. 2g) within a plasmid DNA and accurately transliterated into natural DNA by the E. coli replication machinery in vivo.³⁰⁾ Lastly, short stretches of DNA possessing size-expanded (benzo-fused) Watson–Crick like base pairs, such as an xA : T pair (termed xDNA) (Fig. 2h), can also function as a template for natural DNA replication in vivo.³¹⁾

2.2. Backbone-Modified Genetic Polymers

In addition to nucleobase-modified genetic polymers, pioneering work has investigated synthetic genetic polymers with an artificial backbone. For example, a plasmid containing a few triazole linkages instead of the natural phosphodiester linkages, termed “click-linked DNA” (Fig. 3a), has been accepted as a template by the natural DNA replication and/or mRNA transcription machinery.^32–34) In addition, phosphorothioate (PS) linkage-containing RNA has been shown to raise the mRNA activity due to the increased number of translating ribosomes in prokaryotic translation systems^35,36) (Fig. 3b).

Fig. 3. Structures of Backbone-Modified Elements as Components of Synthetic Genetic Polymer Alternatives to Natural DNA/RNAs

a) Click-linked DNA. b) PS linkage. c) CeNA. d) HNA. e) AraNA. f) (S)-ZNA. Base = A, G, C, T, or U.

With respect to synthetic genetic polymers containing modified sugar moieties, it has been shown that genetic information stored as four consecutive codons can be conveyed to natural DNA in vivo in the form of messages composed of synthetic elements featuring six-membered sugar rings, such as cyclohexenyl nucleic acid (CeNA) (Fig. 3c) and hexitol nucleic acid (HNA) (Fig. 3d), as well as arabinofuranosyl nucleic acid (AraNA)³⁷⁾ (Fig. 3e). More recently, a DNA fragment bearing an acyclic phosphonate backbone, namely (S)-ZNA, has been incorporated into E. coli genetics³⁸⁾ (Fig. 3f).

3. Rewriting the Central Dogma with 4′-ThioDNA and 4′-ThioRNA

As introduced in the previous section, synthetic genetic polymers with a modified sugar-phosphate backbone have been implemented to a limited extent in the hierarchy of the flow of genetic information, specifically at the level of replication or translation. Because the sugar-phosphate backbone architecture is common to both DNA and RNA (except for the 2′-OH group), we have focused our interest on the flow of genetic information from a synthetic DNA-like element with an unnatural backbone to its cognate synthetic RNA-like counterpart, which in turn may possess mRNA activity to encode a phenotype.

In medicinal chemistry, the substitution of a specific atom within a molecule with another atom belonging to the same group of the periodic table is a well-known strategy to develop new therapeutic agents with biological properties similar to those of the parent molecules. Adopting this approach, we have been attempting to rewrite the flow of genetic information using 4′-thioDNA and 4′-thioRNA, which possess a sulfur instead of an oxygen atom in the furanose ring (Fig. 1c). To this end, we examined first whether DNA polymerase can use d^SNTPs to produce 4′-thioDNA, and then whether 4′-thioDNA can be transcribed to produce a cognate 4′-thioRNA.^14–16,19)

3.1. Replication of 4′-ThioDNA Using d^SNTPs and DNA Polymerase

We used primer extension assays to assess the ability of several different DNA polymerases to recognize d^SNTPs as substrates and produce 4′-thioDNA¹⁵⁾ (Fig. 4a). Almost no extension was observed with family A or reverse transcriptase (RT) DNA polymerase (lanes 2–5, 12 and 13), although Tth (lane 4) and M-MLV RT (lane 12) afforded partial extension products (Fig. 4b). The extension efficiency of family Y DNA polymerases (lanes 14 and 15) was better, but a strong band at the position where three nucleotides suggested that the enzyme may have stalled. Overall, the best incorporation efficiency was observed for family B DNA polymerases (lanes 6–11), of which Vent (exo⁻) (lane 6), Deep Vent (exo⁻) (lane 7), Therminator (lane 10), and KOD Dash (lane 11) exhibited notable extension efficiency. The lower activity of Pfu (lane 8) and Tli (lane 9) is probably due to their higher fidelity to 2′-deoxyribonucleoside 5′-triphosphate (dNTPs) due to 3′→5′ exonuclease activity. While the family B DNA polymerases showed the best activity overall, the family Y DNA polymerase Dpo4 (lane 14) exhibited comparable activity when manganese ions were added to the assay (lane 15). Our observations were consistent with another study reporting that family B DNA polymerases tolerate not only nucleotides with modified bases, but also that with modified sugars.^39–50) Thus, family B DNA polymerases are relatively tolerant to modified dNTPs.

Fig. 4. Replication with d^SNTPs to Produce 4′-ThioDNA

Scheme (a) and results (b) of primer extension assay using various DNA polymerases and d^SNTPs. Duplexes of 5′-FITC-labeled primer (0.8 µM) and template (1.0 µM), d^SNTPs (40 µM), and DNA polymerase (0.01–0.4 U/µL) were incubated in reaction buffer (25 µL) at 37 or 74 °C.

We further compared the ability of different family B DNA polymerases to produce 4′-thioDNA by measuring the relative yields of amplicons in PCR experiments under various d^SNTP conditions¹⁵⁾ (Fig. 5). Whereas the amplicon yield was low when Vent, Deep Vent, or Therminator was used with d^SNTPs, both 9°Nm and KOD Dash afforded the desired 4′-thioDNA regardless of the d^SNTP used; in particular, KOD Dash was judged to give the best amplification of 4′-thioDNA.

Fig. 5. Relative Yields of Amplicons in PCR Using Vent, Deep Vent, Therminator, 9°Nm, or KOD Dash DNA Polymerase with all Natural dNTPs or with dNTPs Containing One Type of d^SNTP

The y-axis indicates the replication yield relative to that of KOD Dash DNA polymerase with natural dNTPs.

3.2. Transcription of 4′-ThioDNA to Produce 4′-ThioRNA

Next, we evaluated whether 4′-thioDNA can transfer its genetic information to the corresponding 4′-thioRNA.¹⁹⁾ We performed PCR using KOD Dash DNA polymerase and d^SNTP to generate a series of 4′-thioDNA sequences encoding a T7 RNA promoter, a ribosomal binding (Shine Dalgarno) sequence, and a green florescent protein (GFPuv) coding sequence. We then evaluated T7-mediated transcription using natural DNA or the 4′-thioDNA sequences as a template and triphosphates, of which one was replaced by a r^SNTP. With the natural DNA template, r^SATP, r^SCTP, and r^SUTP were incorporated into transcripts (lanes 2–5, 3.4–59%), albeit at a lower efficiency relative to natural triphosphates (lane 1, 100%); however, r^SGTP was not incorporated (Fig. 6a). The d^SA DNA template generally afforded transcripts in poor yields (lanes 7–10) (Fig. 6b), although the reaction with r^SUTP, in which 4′-thionucleotide pairs (d^SA : ^SU) are formed within the polymerase complex, afforded some transcript (lane 10, 7.6%).

Fig. 6. T7-Mediated Transcription of a 4′-ThioDNA Template in the Presence of r^SNTPs

Shown is gel images of transcripts obtained from a natural (a), d^SA (b), d^SG (c), d^SC (d), or d^ST (e) DNA template. Underlining indicates the r^SNTP that forms a base pair with the 4′-thio units in the 4′-thioDNA template. ^a Relative transcription efficiency (%) was determined by comparing the band intensity of the targeted transcript under each reaction condition to that of transcription using natural DNA with rNTPs (100%).

Furthermore, transcription of the d^SG DNA template in the presence of r^SCTP, which pairs with 4′-thio units in the template (d^SG : r^SC), was comparable to that of the natural template and dNTPs (lane 14, 77% vs. lane 1, 100%) (Fig. 6c). Transcription of templates possessing 4′-thiopyrimidine units, such as d^SC DNA or d^ST DNA, proceeded with r^SCTP (lane 19, 30% and lane 24, 3.0%) (Figs. 6d, e), but at lower efficiency relative to the d^SG DNA template with r^SCTP (lane 14, 77%). Importantly, sequencing analysis confirmed that the 4′-thioDNA was faithfully transcribed to 4′-thioRNA; thus, the genetic information conserved in 4′-thioDNA was successfully transferred to its cognate counterpart 4′-thioRNA.

3.3. Gene Expression from 4′-ThioDNA to 4′-ThioRNA and Eventually to Protein

Our next step in rewriting the central dogma was to try and establish a continuous synthetic flow of genetic information from 4′-thioDNA to 4′-thioRNA and eventually to protein. We therefore conducted a series of one-pot gene expression experiments (Fig. 7a) consisting of a natural or d^SG DNA template encoding GFPuv in a E. coli cell-free transcription–translation system including all natural ribonucleoside 5′-triphosphate (rNTPs), natural rNTPs without rCTP, or natural rNTPs with rCTP replaced with r^SCTP.¹⁹⁾ Fluorescence indicated GFPuv expression from the natural DNA via transcription with natural rNTPs occurred in a test tube (Figs. 7b–d, reaction 1), while no obvious fluorescence was detected in the absence of rCTP (reaction 2). Similarly, GFPuv fluorescence was observed for the d^SG DNA template with natural rNTPs (reaction 3), but no obvious fluorescence was detected in the absence of rCTP (reaction 4). In the case where natural rCTP was replaced by r^SCTP, fluorescence was observed for the d^SG DNA, as well as the natural DNA template (reactions 5 and 6). Thus, gene expression of d^SG DNA occurred via r^SC RNA transcription to give GFPuv (reaction 6), in analogy to the natural genetic system (reaction 1), although the gene expression efficiency was lower. In summary, we succeeded in rewriting the central dogma, whereby the genetic information stored in 4′-thioDNA was transferred to 4′-thioRNA, which in turn exerted mRNA activity to express protein. These results underscore the feasibility of controlling life by substances other than DNA and RNA nucleotides.

Fig. 7. Rewriting the Central Dogma with 4′-ThioDNA and 4′-ThioRNA in a Test Tube

a) Protocol for one-pot gene expression experiments using natural DNA or d^SG DNA encoding GFPuv in an E. coli cell-free system with either natural rNTPs or with rCTP removed or replaced by r^SCTP. b) Template and nucleoside 5′-triphosphate combinations in each reaction. c) Confirmation of GFPuv expression by fluorescence image. d) Comparison of gene expression efficiency by Western blotting analysis. The band intensity of reaction 1 was set to 100%. Error bars indicate the standard deviations of three independent experiments. ^a n.d. = not detected.

To further develop this concept, we explored whether we could rewrite the gene expression system using 4′-thioDNA and 4′-thioRNA in an artificial cell (Fig. 8). The artificial cell comprised a lipid bilayer vesicle formed by the thin-film hydration method using 1,2-dioleoyl-sn-glycero-3-phosphocholine in a solution containing E. coli cell-free transcription–translation machinery⁵¹⁾ with r^SCTP instead of rCTP and the d^SG DNA template. After vesicle formation, proteinase K was added to quench any extracellularly expressed GFPuv. As a result, fluorescence was observed inside the vesicle, demonstrating that the genetic information stored in d^SG DNA was transcribed into r^SC RNA, followed by production of GFPuv based on the genetic information in the transcribed r^SC RNA (Fig. 8b), mirroring the gene expression occurring with natural DNA and rNTPs (Fig. 8a). When r^SCTP was omitted from the artificial cell, no expression of GFPuv from d^SG DNA was observed due to the missing r^SCTP building block for r^SC RNA transcription; however, formation of the lipid bilayer vesicle was confirmed by Nile Red staining (Fig. 8c). Thus, these findings demonstrate the feasibility of gene expression of 4′-thioDNA via 4′-thioRNA transcription in an artificial cell.

Fig. 8. Gene Expression from d^SG DNA via Transcription to r^SC DNA in an Artificial

Cells comprise E. coli gene expression machinery with r^SCTP instead of rCTP. Proteinase K was added to quench fluorescence from products of gene expression outside the vesicles. a) Control with natural DNA as the genetic template and rNTPs. b) d^SG DNA with r^SCTP instead of rCTP. c) d^SG DNA without r^SCTP as a r^SC RNA building block (left). The lipid layer was stained with Nile Red to confirm vesicle formation (right).

Of note, 4′-thioDNA and 4′-thioRNA are known to be significantly resistant to enzymatic degradation,^4,8,11–13) and this enhanced stability may be advantageous for gene expression in an artificial cell under certain conditions.

4. Conclusion and Outlook towards Therapeutic Applications

Synthetic genetic polymers that can be written into the central dogma of molecular biology are becoming progressively more plausible as the number and diversity of nucleic acid chemistries increase.³⁾ Indeed, some of these elements are already advancing as new therapeutic modalities in the field of drug discovery.^52–58) The 4′-thionucleic acids discussed above are a prominent example of such elements; indeed, we have succeeded in creating genetic systems composed of 4′-thionucleic acids for different therapeutic applications, including the development of gene vectors composed of 4′-thioDNA (named an “intelligent RNA expression device” or iRed)^17,18,20) and 4′-thioRNA aptamers.^5,10) Furthermore, several synthetic genetic polymers are showing promise in addressing the shortcomings that limit the utility of natural nucleic acids in therapeutic contexts. For example, some genetic polymers have been proven to attenuate¹⁷⁾ or enhance⁵⁹⁾ innate immune induction via immune receptors that generally recognize natural DNA and/or RNA. It can be anticipated that future advances in this field will lead to rapid progress in modern drug discovery that will further challenge the central dogma of molecular biology.

Acknowledgments

We wish to thank all our colleagues, especially Prof. Dr. Akira Matsuda, Ms. Ayako Matsuo, Dr. Naonori Inoue, Dr. Hideto Maruyama, and Mr. Takamitsu Kojima. This work was financially supported in part by JSPS KAKENHI Grant Numbers 21H02606 and 21K19052. N.S.T. thanks the research program for the development of intelligent Tokushima artificial exosome (iTEX) from Tokushima University.

Conflict of Interest

The authors declare no conflict of interest.

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