Evolution of abiotic cubane chemistries in a nucleic acid aptamer allows selective recognition of a malaria biomarker Yee-Wai Cheunga,1, Pascal Röthlisbergerb,1, Ariel E. Mechalyc, Patrick Weberc, Fabienne Levi-Acobasb, Young Loa, Alvin W. C. Wonga, Andrew B. Kinghorna, Ahmed Haouzc, G. Paul Savaged, Marcel Hollensteinb,2,3, and Julian A. Tannera,2,3 aSchool of Biomedical Sciences, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong, China; bInstitut Pasteur, Laboratory for Bioorganic Chemistry of Nucleic Acids, Department of Structural Biology and Chemistry, CNRS UMR-3523, 75015 Paris, France; cCrystallography Platform-C2RT, Department of Structural Biology and Chemistry, CNRS UMR-3528, Institut Pasteur, 75015 Paris, France; and dCSIRO Manufacturing, Clayton, VIC 3168, Australia Edited by Jack W. Szostak, Massachusetts General Hospital, Boston, MA, and approved May 31, 2020 (received for review February 20, 2020) Nucleic acid aptamers selected through systematic evolution of aptamers (18, 20–23). Other approaches include aptamers with ligands by exponential enrichment (SELEX) fold into exquisite artificially expanded genetic information systems going beyond globular structures in complex with protein targets with diverse the simple four building blocks of natural nucleic acids (24), even translational applications. Varying the chemistry of nucleotides most recently to the eight-building-block Hachimoji system (25). allows evolution of nonnatural nucleic acids, but the extent to Nucleobases have also been modified but have often been re- which exotic chemistries can be integrated into a SELEX selection stricted to mimics of amino acid side chains (13, 26, 27) or small to evolve nonnatural macromolecular binding interfaces is unclear. hydrophobic aromatic moieties (28, 29). Perhaps the most im- Here, we report the identification of a cubane-modified aptamer pressive demonstration of translational cross-proteome aptamer (cubamer) against the malaria biomarker Plasmodium vivax lactate specificity are the SOMAmers (slow off-rate modified aptamers) dehydrogenase (PvLDH). The crystal structure of the complex re- (30), which enabled quantification of over 3,000 plasma proteins veals an unprecedented binding mechanism involving a multicu- CHEMISTRY bane cluster within a hydrophobic pocket. The binding interaction to create a genomic atlas of the human plasma proteome (31). is further stabilized through hydrogen bonding via cubyl hydro- Such SOMAmers include a simple modification where instead of gens, previously unobserved in macromolecular binding inter- dT they bear dU functionalized at the 5-position of the nucleo- faces. This binding mechanism allows discriminatory recognition base with protein-like benzyl/2-napthyl/3-indolyl-carboxamide of P. vivax over Plasmodium falciparum lactate dehydrogenase, that confer some hydrophobic and/or base stacking character to thereby distinguishing these highly conserved malaria biomarkers for diagnostic applications. Together, our data demonstrate that Significance SELEX can be used to evolve exotic nucleic acids bearing chemical functional groups which enable remarkable binding mechanisms We report the identification of a cubane-modified aptamer which have never been observed in biology. Extending to other (cubamer) against the malaria biomarker Plasmodium vivax exotic chemistries will open a myriad of possibilities for functional lactate dehydrogenase (PvLDH). The cubamer contains the nucleic acids. benzene isostere cubane, which is entirely alien to biology. The crystal structure of the cubamer–protein complex reveals a modified aptamer | SELEX | X-ray crystallography | cubane | malaria binding mechanism involving the formation of an unprece- diagnosis dented cubane pocket and an unusual C–H···O hydrogen bond. Importantly, the cubamer is capable of distinguishing the irected evolution of nucleic acids through the iterative PvLDH from the Plasmodium falciparum LDH despite a very Dprocess of SELEX (1, 2) (selective evolution of ligands by high sequence homology, which is impossible for unmodified exponential enrichment) provides routes to aptamers with ex- aptamers. Finally, we have used the cubamer to detect PvLDH quisite structures that cannot be otherwise imagined or designed in a mimetic clinical situation. This approach blending medicinal (3). Even using the simple natural nucleotides of DNA and RNA chemistry and Darwinian evolution can easily be extended to one can select and evolve aptamers with nanomolar and even other nonnatural, exotic functional groups. picomolar binding affinities and surprising discriminatory speci- ficity (4). Such aptamers are finding applications in therapeutics Author contributions: Y.-W.C., P.R., M.H., and J.A.T. designed research; Y.-W.C., P.R., A.E.M., P.W., F.L.-A., Y.L., A.W.C.W., A.B.K., and A.H. performed research; G.P.S. contrib- (5), diagnostics (6), biosensing (7), and nanotechnology (8) and uted new reagents/analytic tools; Y.-W.C., P.R., A.E.M., F.L.-A., Y.L., A.B.K., A.H., M.H., and can display numerous advantages relative to antibodies (9). J.A.T. analyzed data; and Y.-W.C., P.R., M.H., and J.A.T. wrote the paper. The chemistries of canonical DNA and RNA can be limiting Competing interest statement: J.A.T. is a scientific advisor for Zio Health, and J.A.T. and when one compares them to antibodies (10). Postselection A.B.K. are founders of Jushan Bio. chemical modification can aid in vivo stability and residence time This article is a PNAS Direct Submission. (11, 12) and can facilitate binding to problematic targets such as Published under the PNAS license. – glycoproteins or proteins with low isoelectric points (13 15). Data deposition: The crystal structure of the cubamer–target complex has been deposited However, these post-SELEX approaches often come at the ex- in the Protein Data Bank, http://www.wwpdb.org/ (PDB ID code 6TXR). pense of a loss of binding affinity and specificity. In contrast, 1Y.-W.C. and P.R. contributed equally to this work. — 2 preselection chemical modification where modified nucleo- M.H. and J.A.T. contributed equally to this work. — tides are directly included in the SELEX process truly opens 3To whom correspondence may be addressed. Email: [email protected] or up directed evolution to extending the repertoire of aptamer- [email protected]. mediated molecular recognition (16–19). Many chemical modi- This article contains supporting information online at https://www.pnas.org/lookup/suppl/ fications have been directed at the level of the sugar-phosphate doi:10.1073/pnas.2003267117/-/DCSupplemental. backbone in order to improve the nuclease resistance of www.pnas.org/cgi/doi/10.1073/pnas.2003267117 PNAS Latest Articles | 1of9 Downloaded by guest on September 29, 2021 the nucleic acid (10). New, engineered polymerases are not re- Investigating Compatibility of dUCTP with Primer Extension and PCR. quired to cope with unnatural chemistry introduced at position 5 We next asked whether dUCTP 11 (Fig. 1B) would be compatible of pyrimidines (32). Base modified nucleotides have also been with enzymatic DNA synthesis under primer extension (PEX) proposed as elements in an RNA world to promote the catalytic reaction and PCR conditions. We used the 15-nucleotide-long, 5′ activity of RNA-based catalysts (33). Alternatively, click chem- 6-FAM (fluorescein)-labeled primer P1 annealed to template T1 istry with alkyne-modified nucleotides can be used to select for (see SI Appendix for sequence composition) then investigated the nucleobase-modified aptamers (34). Through such approaches, ability of various polymerases to perform a PEX reaction with a nucleobase-modified aptamers with nonnatural chemistries be- mixture of either four natural triphosphates or three natural C come accessible. One can then take advantage of the exponential triphosphates together with dU TP replacing thymidine 5′- enrichment and mutation enabled by the PCR to allow Dar- triphosphate (dTTP) (SI Appendix, Fig. S20). This analysis C winian molecular evolution, yet inclusion of more exotic un- revealed that most polymerases accepted dU TP as a substrate natural functional groups allows evolutionary experiments well and the primer was extended by 16 nucleotides (accounting for beyond the confined chemistries familiar of biology. the incorporation of four modified nucleotides). When a longer Here, we present an effort to stretch the idea of nonnatural template was used (the 79-mer template T2) which would re- P2 chemistry within an evolutionary nucleic acid aptamer selection quire extension of primer by 60 nucleotides a clear by applying the in vitro evolutionary technique of SELEX to higher-molecular-weight product is formed in the reaction with C SI macromolecular chemistries entirely alien to biology. We per- dU TP when compared to that with natural triphosphates ( Appendix form an aptamer selection incorporating unusual cubane- , Figs. S21 and S22). Several polymerases including exo− Bst modified nucleotides, presenting structural data, mechanistic Therminator, Vent ( ), Deep Vent, Taq, , Klenow, Q5 determination, and diagnostic application of cubane-modified DNA polymerase, and phi29 were all capable of forming the exo− nucleic acid aptamers, hereafter called cubamers. The extraor- expected cubane-modified product. However, Vent ( ) appeared to most consistently produce highest-yield product (SI dinary platonic solid cubanes have intrigued chemists for de- Appendix cades (35–37) and are now important functional groups in a wide