Editorial Feature

Sequence-Defined Synthetic Polymers and the New Class of Army Therapeutics

Biomaterial - Biopolymer - Abstract Illustration 

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Sequence-defined synthetic polymers are a promising new class of materials that has found its potential application in a wide range of fields, including nanotechnology and biomedicine. Unfortunately, the traditional synthesis of these polymers is associated with various limitations. As a result, a group of researchers supported by the United States Army recently discovered how flexizymes could be used to improve this polymerization process.

What are Sequence-Defined Polymers (SDPs)?

Both SDPs and sequence-defined oligomers (SDOs) are chain molecules comprised of a specific order of monomer units. Within each chain molecule of SDOs and SDPs exists a monomeric sequence that is identical to every other molecule and functions as the blueprint for any cellular function in biological organisms. When synthesized by chemical means, both SDOs and SDPs have been applied to nanoelectronics devices, molecular recognition systems, high-density information systems, biomedical treatments, and much more.

Traditional Methods of Synthesizing Sequence-Defined Polymers

Within the laboratory, the generation of sequence-defined biological polymers has been achieved by transforming RNA, DNA, and polypeptides to molecules that exhibit a wide range of binding and catalytic properties. Some of the most common clinical methods used for the synthesis of SDPs include polymerase chain reaction (PCR), in vitro protein expression, or solid-phase synthesis (SPS).

Despite the fact that several different laboratory techniques are available for this purpose, the chemical synthesis of SDPs has been limited to the synthesis of nucleic acid and polypeptide analogs by polymerases and ribosomes. Because polymerases can only utilize mononucleotides as their substrates, the possible diversity of synthetic polymers created by these enzymes is significantly hindered. In addition to overcoming the issue of limited polymer diversity, researchers are also interested in developing alternative strategies that are both scalable and sustainable.

Manipulating Natural Polymerization

In an effort to expand the number of biopolymers available for public use, researchers have remained curious as to how natural polymerization processes, such as that which is achieved by the Escherichia coli (E. coli) protein biosynthesis system, can do just that. Such efforts work under the belief that the incorporation of diverse ribosomal monomers into a single polymeric compound will lead to the development of a wide range of novel therapeutics, enzymes, and materials.

To date, the natural ribosome has been used as a template for the elongation of polymer chains through the selective addition of numerous chemical substrates, some of which include hydroxy acids, peptides, oligomeric foldamer-peptide hybrids, and non-amino carboxylic acids. This incorporation is first achieved by the covalent attachment of these monomers to transfer RNAs (tRNAs), which creates aminoacyl-tRNA substrates. Typically, this process is achieved by a process known as chemical aminoacylation, which involves the synthesis of 5’-phospho-2’-deoxyribocytidylylriboadenosine (pdCpA) dinucleotide, which is then followed by the coupling of the amino-acid substrate to an ester. Finally, enzyme ligation with a truncated tRNA completes the synthesis of such noncanonical aminoacyl-tRNAs.

The Rise of Flexizymes

Flexizymes (Fxs) are a type of ribozyme that has been derived from a tRNA synthetase. During the biological process of translation, tRNA synthetases catalyze a process known as aminoacylation, which occurs when an anticodon of the tRNA strand is charged with its matching amino acid. In nature, this process plays a key role in determining the genetic code. Therefore, the researchers who originally developed Fxs were hoping to create a unique and flexible ribozyme that can selectively act as an aminoacylation catalyst for the manipulation of genetic sequences.

In addition to allowing researchers to create a second genetic code within the laboratory, Fxs have also allowed scientists to create modern experimental surrogates that closely resemble ancient genetic codes to improve the understanding of the origin and evolution of RNA. To date, three distinct types of Fxs have been developed to recognize specific combinations of a substrate-activating group, which includes eFx, dFx, and aFx. Whereas eFxs acylates tRNA with cyanomethyl ester (CME)-activated acids, dFxs instead recognize dinitrobenzyle ester (DNBE)-activated non-aryl acids and aFxs recognize (2-aminoethyl) amidocarboxybenzyl thioester (ABT) leaving groups.

Flexizymes for SDP Production

A recent study conducted by a group of researchers supported by the United States Army utilized Fx technology to expand the current range of chemical substrates available for template-guided polymerization. To this end, a total of 37 phenylalanine derivatives, benzoic acid derivatives, heteroaromatic monomers, and aliphatic monomers were synthesized and used as substrates for potential Fx charging. Of the total 37 substrates investigated in this study, the researchers found that the Fxs were capable of charging 32 substrates to tRNA molecules.

Notably, several guiding principles were established during the course of these Fx-catalyzed reactions. For example, the favorability of electron-deficient substrates that also exhibited a lower level of steric hindrance around the carbonyl group was confirmed to show higher compatibility with the Fx system when compared to electron-rich substrates. Additionally, certain chemical groups that are bulkier in nature were found to be less tolerated at the acylation site.

Once the novel tRNA-monomers were created by Fx integration methods, their ribosomal polymerization capabilities were assessed. The utility of these peptide hybrids in bioconjugation was then determined through the initiation of an imine-forming reaction between the aldehyde and hydrazine of the tRNA monomer. This experiment confirmed the ability of Fxs to ultimately create SDPs that are applicable to the creation of innovative high-performance materials and therapeutic agents. 

References and Further Reading

Leibfarth, F. A., Johnson, J. A., & Jamison, T. F. (2015). Scalable synthesis of sequence-defined unimolecular macromolecules by Flow-IEG. PNAS 112(34); 10617-10622. DOI: 10.1073/pnas.1508599112.

Yu, H., Li, S., Schqieter, K. E., Liu, Y., Sun, B., Moore, J. S., & Shroeder, C. M. (2020). Charge Transport in Sequence-Defined Conjugated Oligomers. Journal of the American Chemical Society. DOI: 10.1021/jacs.0c00043.

Morimoto, J., Hayashi, Y., Iwasaki, K., & Suga, H. (2011). Flexizymes: Their Evolutionary history of the Origin of Catalytic Function. Accounts of Chemical Research 44(12); 1359-1368. DOI: 10.1021/ar2000953.

Lee, J., Shcwieter, K. E., Watkins, A. M., Kim, D. S., Yu, H., Schwarz, K. J., et al. (2019). Expanding the limits of the second genetic code with ribozymes. Nature Communications 10(5097). DOI: 10.1038/s41467-019-12916-w.

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Benedette Cuffari

Written by

Benedette Cuffari

After completing her Bachelor of Science in Toxicology with two minors in Spanish and Chemistry in 2016, Benedette continued her studies to complete her Master of Science in Toxicology in May of 2018. During graduate school, Benedette investigated the dermatotoxicity of mechlorethamine and bendamustine, which are two nitrogen mustard alkylating agents that are currently used in anticancer therapy.


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