Monitoring SARS-CoV-2 Nsp13 helicase binding activity using expanded genetic code techniques
Eryn Lundrigan, Christine Hum, Nadine Ahmed, John Paul Pezacki
RSC Chemical Biology, doi:10.1039/d4cb00230j
The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) non-structural protein 13 (Nsp13) helicase is a multi-functional protein that can unwind dsDNA and dsRNA in an NTP-dependent manner. Given that this viral helicase is essential for viral replication and highly conserved among coronaviruses, a thorough understanding of the helicase's unwinding and binding activity may allow for the development of more effective pan-coronavirus therapeutics. Herein, we describe the use of genetic code expansion techniques to site-specifically incorporate the non-canonical amino acid (ncAA) p-azido-L-phenylalanine (AzF) into Nsp13 for fluorescent labelling of the enzyme with a conjugated Cy5 fluorophore. This Cy5labelled Nsp13-AzF can then be used in Fo ¨rster resonance energy transfer (FRET) experiments to investigate the dynamics of enzyme translocation on its substrate during binding and unwinding. Five sites (F81, F90, Y205, Y246, and Y253) were identified for AzF incorporation in Nsp13 and assessed for fluorescent labelling efficiency. The incorporation of AzF was confirmed to not interfere with the unwinding activity of the helicase. Subsequently, FRET-based binding assays were conducted to monitor the binding of Cy5-labelled Nsp13-AzF constructs to a series of fluorescently-labelled nucleic acid substrates in a distance-dependent manner. Overall, this approach not only allows for the direct monitoring of Nsp13's binding activity on its substrate, it may also introduce a novel method to screen for compounds that can inhibit this essential enzymatic activity during viral replication.
Author contributions C. Hum performed experimental work, data collection, analysis and writing the original draft. N. Ahmed contributed to conceptualization of the study. E. Lundrigan performed experimental work, data collection, and analysis and contributed to the writing and revising at the stage of review. J. P. Pezacki
Conflicts of interest There are no conflicts to declare.
References
Ablenas, Gidi, Powdrill, Hepatitis C Virus Helicase Binding Activity Monitored through Site-Specific Labeling Using an Expanded Genetic Code, ACS Infect. Dis,
doi:10.1021/acsinfecdis.9b00220Adedeji, Singh, Calcaterra, Severe Acute Respiratory Syndrome Coronavirus Replication Inhibitor That Interferes with the Nucleic Acid Unwinding of the Viral Helicase, Antimicrob. Agents Chemother,
doi:10.1128/AAC.00957-12Berta, Badaoui, Martino, Modelling the active SARS-CoV-2 helicase complex as a basis for structurebased inhibitor design, Chem. Sci,
doi:10.1039/D1SC02775AChen, Malone, Llewellyn, Structural Basis for Helicase-Polymerase Coupling in the SARS-CoV-2 Replication-Transcription Complex, Cell,
doi:10.1016/J.CELL.2020.07.033Chin, Santoro, Martin, King, Wang et al., Addition of p-azido-L-phenylalanine to the genetic code of Escherichia coli, J. Am. Chem. Soc,
doi:10.1021/ja027007wChin, Santoro, Martin, King, Wang et al., Addition of p-azido-L-phenylalanine to the genetic code of Escherichia coli, J. Am. Chem. Soc,
doi:10.1021/ja027007wGurung, In silico structure modelling of SARS-CoV-2 Nsp13 helicase and Nsp14 and repurposing of FDA approved antiviral drugs as dual inhibitors, Gene Rep,
doi:10.1016/j.genrep.2020.100860Herbert, Poptsova, Z-RNA and the Flipside of the SARS Nsp13 Helicase: Is There a Role for Flipons in Coronavirus-Induced Pathology?, Front. Immunol,
doi:10.3389/fimmu.2022.912717Hoogenboom, Thiol-Yne Chemistry: A Powerful Tool for Creating Highly Functional Materials, Angew. Chem., Int. Ed,
doi:10.1002/anie.201000401Hwang, Myong, Protein induced fluorescence enhancement (PIFE) for probing protein-nucleic acid interactions, Chem. Soc. Rev,
doi:10.1039/c3cs60201jIqbal, Wang, Thompson, Lilley, Norman, The structure of cyanine 5 terminally attached to double-stranded DNA: Implications for FRET studies, Biochemistry,
doi:10.1021/bi800773fIvanov, Thiel, Dobbe, Van Der Meer, Snijder et al., Multiple Enzymatic Activities Associated with Severe Acute Respiratory Syndrome Coronavirus Helicase, J. Virol,
doi:10.1128/jvi.78.11.5619-5632.2004Jakob, Gust, Grohmann, Evaluation and optimisation of unnatural amino acid incorporation and bioorthogonal bioconjugation for site-specific fluorescent labelling of proteins expressed in mammalian cells, Biochem. Biophys. Rep,
doi:10.1016/j.bbrep.2018.10.011Jang, Jeong, Kang, Sp, Yang et al., A high ATP concentration enhances the cooperative translocation of the SARS coronavirus helicase nsP13 in the unwinding of duplex RNA, Sci. Rep,
doi:10.1038/s41598-020-61432-1Jia, Yan, Ren, Delicate structural coordination of the Severe Acute Respiratory Syndrome coronavirus Nsp13 upon ATP hydrolysis, Nucleic Acids Res,
doi:10.1093/nar/gkz409Lang, Chin, Cellular incorporation of unnatural amino acids and bioorthogonal Labeling of Proteins, Chem. Rev,
doi:10.1021/cr400355wMartin, Li, Parvangada, Genetic conservation of SARS-CoV-2 RNA replication complex in globally circulating isolates and recently emerged variants from humans and minks suggests minimal pre-existing resistance to remdesivir, Antiviral Res,
doi:10.1016/j.antiviral.2021.105033Marx, Mickolajczyk, Craig, Thomas, Pfeffer et al., Observing inhibition of the SARS-CoV-2 helicase at single-nucleotide resolution, Nucleic Acids Res,
doi:10.1093/nar/gkad660Mathieu, Ritchie, Rode ´s-Guirao, Coronavirus Pandemic (COVID-19). Our World in Data
Newman, Douangamath, Yadzani, Structure, mechanism and crystallographic fragment screening of the SARS-CoV-2 NSP13 helicase, Nat. Commun,
doi:10.1038/s41467-021-25166-6Pak, Adegboye, Adekunle, Rahman, Mcbryde et al., Economic Consequences of the COVID-19 Outbreak: the Need for Epidemic Preparedness, Front. Public Health,
doi:10.3389/fpubh.2020.00241Perez-Lemus, Mene ´ndez, Alvarado, Byle ´hn, De, Toward wide-spectrum antivirals against coronaviruses: Molecular characterization of SARS-CoV-2 NSP13 helicase inhibitors, Sci. Adv,
doi:10.1126/sciadv.abj4526Rosas-Lemus, Minasov, Shuvalova, Highresolution structures of the SARS-CoV-2 2 0 -O-methyltransferase reveal strategies for structure-based inhibitor design, Sci. Signaling,
doi:10.1126/scisignal.abe1202Seybert, Hegyi, Siddell, Ziebuhr, The human coronavirus 229E superfamily 1 helicase has RNA and DNA duplex-unwinding activities with 5 0 -to-3 0 polarity, RNA,
doi:10.1017/s1355838200000728Shrestha, Jenei, Nagy, Vereb, Szo ¨ll+ osi, Understanding FRET as a research tool for cellular studies, Int. J. Mol. Sci,
doi:10.3390/ijms16046718Tanner, Watt, Chai, The severe acute respiratory syndrome (SARS) coronavirus NTPasefhelicase belongs to a distinct class of 5 0 to 3 0 viral helicases, J. Biol. Chem,
doi:10.1074/jbc.c300328200Wang, Sachdeva, Cox, Optimized orthogonal translation of unnatural amino acids enables spontaneous protein double-labelling and FRET, Nat. Chem,
doi:10.1038/nchem.1919Wilamowski, Sherrell, Minasov, 2 0 -O methylation of RNA cap in SARS-CoV-2 captured by serial crystallography, Proc. Natl. Acad. Sci. U. S. A,
doi:10.1073/pnas.2100170118Yan, Zheng, Zeng, He, Cheng, Structural biology of SARS-CoV-2: open the door for novel therapies, Signal Transduction Targeted Ther,
doi:10.1038/s41392-022-00884-5Yu, Lee, Lee, Identification of myricetin and scutellarein as novel chemical inhibitors of the SARS coronavirus helicase, nsP13, Bioorg. Med. Chem. Lett,
doi:10.1016/J.BMCL.2012.04.081Zander, Holzmeister, Klose, Tinnefeld, Grohmann, Single-molecule FRET supports the two-state model of Argonaute action, RNA Biol,
doi:10.4161/RNA.27446Zeng, Weissmann, Bertolin, Identifying SARS-CoV-2 antiviral compounds by screening for small molecule inhibitors of nsp13 helicase, Biochem. J,
doi:10.1042/bcj20210201Zhang, Li, Cowley, The nsp1, nsp13, and M Proteins Contribute to the Hepatotropism of Murine Coronavirus JHM.WU, J. Virol,
doi:10.1128/jvi.03535-14DOI record:
{
"DOI": "10.1039/d4cb00230j",
"ISSN": [
"2633-0679"
],
"URL": "http://dx.doi.org/10.1039/d4cb00230j",
"abstract": "<jats:p>The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) non-structural protein 13 (Nsp13) helicase is a multi-functional protein that can unwind dsDNA and dsRNA in an NTP-dependent manner.</jats:p>",
"author": [
{
"ORCID": "https://orcid.org/0000-0001-5896-9752",
"affiliation": [
{
"name": "Department of Chemistry and Biomolecular Sciences, University of Ottawa, Ottawa, Ontario K1N 6N5, Canada"
}
],
"authenticated-orcid": false,
"family": "Lundrigan",
"given": "Eryn",
"sequence": "first"
},
{
"affiliation": [
{
"name": "Department of Chemistry and Biomolecular Sciences, University of Ottawa, Ottawa, Ontario K1N 6N5, Canada"
}
],
"family": "Hum",
"given": "Christine",
"sequence": "additional"
},
{
"affiliation": [
{
"name": "Department of Chemistry and Biomolecular Sciences, University of Ottawa, Ottawa, Ontario K1N 6N5, Canada"
}
],
"family": "Ahmed",
"given": "Nadine",
"sequence": "additional"
},
{
"ORCID": "https://orcid.org/0000-0002-4233-8945",
"affiliation": [
{
"name": "Department of Chemistry and Biomolecular Sciences, University of Ottawa, Ottawa, Ontario K1N 6N5, Canada"
}
],
"authenticated-orcid": false,
"family": "Pezacki",
"given": "John Paul",
"sequence": "additional"
}
],
"container-title": "RSC Chemical Biology",
"container-title-short": "RSC Chem. Biol.",
"content-domain": {
"crossmark-restriction": true,
"domain": [
"rsc.org"
]
},
"created": {
"date-parts": [
[
2025,
4,
21
]
],
"date-time": "2025-04-21T17:41:06Z",
"timestamp": 1745257266000
},
"deposited": {
"date-parts": [
[
2025,
4,
29
]
],
"date-time": "2025-04-29T08:31:19Z",
"timestamp": 1745915479000
},
"funder": [
{
"DOI": "10.13039/501100000038",
"award": [
"210719"
],
"doi-asserted-by": "publisher",
"id": [
{
"asserted-by": "publisher",
"id": "10.13039/501100000038",
"id-type": "DOI"
}
],
"name": "Natural Sciences and Engineering Research Council of Canada"
}
],
"indexed": {
"date-parts": [
[
2025,
4,
29
]
],
"date-time": "2025-04-29T09:10:09Z",
"timestamp": 1745917809898,
"version": "3.40.4"
},
"is-referenced-by-count": 0,
"issued": {
"date-parts": [
[
2025
]
]
},
"language": "en",
"license": [
{
"URL": "http://creativecommons.org/licenses/by-nc/3.0/",
"content-version": "vor",
"delay-in-days": 110,
"start": {
"date-parts": [
[
2025,
4,
21
]
],
"date-time": "2025-04-21T00:00:00Z",
"timestamp": 1745193600000
}
}
],
"link": [
{
"URL": "http://pubs.rsc.org/en/content/articlepdf/2025/CB/D4CB00230J",
"content-type": "unspecified",
"content-version": "vor",
"intended-application": "similarity-checking"
}
],
"member": "292",
"original-title": [],
"prefix": "10.1039",
"published": {
"date-parts": [
[
2025
]
]
},
"published-online": {
"date-parts": [
[
2025
]
]
},
"publisher": "Royal Society of Chemistry (RSC)",
"reference": [
{
"DOI": "10.1038/s41586-020-2008-3",
"author": "Wu",
"doi-asserted-by": "publisher",
"first-page": "265",
"issue": "7798",
"journal-title": "Nature",
"key": "D4CB00230J/cit1/1",
"volume": "579",
"year": "2020"
},
{
"DOI": "10.3389/fpubh.2020.00241",
"author": "Pak",
"doi-asserted-by": "publisher",
"first-page": "241",
"journal-title": "Front. Public Health",
"key": "D4CB00230J/cit2/1",
"volume": "8",
"year": "2020"
},
{
"DOI": "10.1371/journal.ppat.1008536",
"author": "Tang",
"doi-asserted-by": "publisher",
"first-page": "e1008536",
"issue": "5",
"journal-title": "PLoS Pathog.",
"key": "D4CB00230J/cit3/1",
"volume": "16",
"year": "2020"
},
{
"DOI": "10.1016/S1473-3099(20)30120-1",
"author": "Dong",
"doi-asserted-by": "publisher",
"key": "D4CB00230J/cit4/1",
"unstructured": "E.Dong , H.Du and L.Gardner An interactive web-based dashboard to track COVID-19 in real time . Published online 2020 10.1016/S1473-3099(20)30120-1",
"year": "2020"
},
{
"author": "Mathieu",
"key": "D4CB00230J/cit5/1",
"unstructured": "E.Mathieu , H.Ritchie , L.Rodés-Guirao , et al. Coronavirus Pandemic (COVID-19) . Our World in Data . Published online March 5, 2020. Accessed January 15, 2023. https://ourworldindata.org/coronavirus"
},
{
"DOI": "10.1128/jvi.03535-14",
"author": "Zhang",
"doi-asserted-by": "publisher",
"first-page": "3598",
"issue": "7",
"journal-title": "J. Virol.",
"key": "D4CB00230J/cit6/1",
"volume": "89",
"year": "2015"
},
{
"DOI": "10.1016/j.virusres.2014.12.001",
"author": "Lehmann",
"doi-asserted-by": "publisher",
"first-page": "12",
"journal-title": "Virus Res.",
"key": "D4CB00230J/cit7/1",
"volume": "202",
"year": "2015"
},
{
"DOI": "10.1016/j.genrep.2020.100860",
"author": "Gurung",
"doi-asserted-by": "publisher",
"first-page": "100860",
"journal-title": "Gene Rep.",
"key": "D4CB00230J/cit8/1",
"volume": "21",
"year": "2020"
},
{
"DOI": "10.1038/s41598-020-61432-1",
"author": "Jang",
"doi-asserted-by": "publisher",
"first-page": "4481",
"journal-title": "Sci. Rep.",
"key": "D4CB00230J/cit9/1",
"volume": "10",
"year": "2020"
},
{
"DOI": "10.1038/s41467-021-25166-6",
"author": "Newman",
"doi-asserted-by": "publisher",
"first-page": "1",
"issue": "1",
"journal-title": "Nat. Commun.",
"key": "D4CB00230J/cit10/1",
"volume": "12",
"year": "2021"
},
{
"DOI": "10.1016/j.bpj.2020.11.2276",
"author": "Mickolajczyk",
"doi-asserted-by": "publisher",
"first-page": "1020",
"issue": "6",
"journal-title": "Biophys. J.",
"key": "D4CB00230J/cit11/1",
"volume": "120",
"year": "2021"
},
{
"DOI": "10.1074/jbc.c300328200",
"author": "Tanner",
"doi-asserted-by": "publisher",
"first-page": "39578",
"issue": "41",
"journal-title": "J. Biol. Chem.",
"key": "D4CB00230J/cit12/1",
"volume": "278",
"year": "2003"
},
{
"DOI": "10.1128/jvi.78.11.5619-5632.2004",
"author": "Ivanov",
"doi-asserted-by": "publisher",
"first-page": "5619",
"issue": "11",
"journal-title": "J. Virol.",
"key": "D4CB00230J/cit13/1",
"volume": "78",
"year": "2004"
},
{
"DOI": "10.1371/journal.pone.0036521",
"author": "Adedeji",
"doi-asserted-by": "publisher",
"first-page": "e36521",
"issue": "5",
"journal-title": "PLoS One",
"key": "D4CB00230J/cit14/1",
"volume": "7",
"year": "2012"
},
{
"DOI": "10.1073/pnas.2100170118",
"author": "Wilamowski",
"doi-asserted-by": "publisher",
"first-page": "e2100170118",
"issue": "21",
"journal-title": "Proc. Natl. Acad. Sci. U. S. A.",
"key": "D4CB00230J/cit15/1",
"volume": "118",
"year": "2021"
},
{
"DOI": "10.1126/scisignal.abe1202",
"author": "Rosas-Lemus",
"doi-asserted-by": "publisher",
"first-page": "1202",
"issue": "651",
"journal-title": "Sci. Signaling",
"key": "D4CB00230J/cit16/1",
"volume": "13",
"year": "2020"
},
{
"DOI": "10.1038/s41586-022-05185-z",
"author": "Park",
"doi-asserted-by": "publisher",
"first-page": "793",
"issue": "7928",
"journal-title": "Nature",
"key": "D4CB00230J/cit17/1",
"volume": "609",
"year": "2022"
},
{
"DOI": "10.1038/s41467-020-17496-8",
"author": "Viswanathan",
"doi-asserted-by": "publisher",
"first-page": "1",
"issue": "1",
"journal-title": "Nat. Commun.",
"key": "D4CB00230J/cit18/1",
"volume": "11",
"year": "2020"
},
{
"DOI": "10.1038/s41392-022-00884-5",
"author": "Yan",
"doi-asserted-by": "publisher",
"first-page": "1",
"issue": "1",
"journal-title": "Signal Transduction Targeted Ther.",
"key": "D4CB00230J/cit19/1",
"volume": "7",
"year": "2022"
},
{
"DOI": "10.1016/J.CELL.2020.07.033",
"author": "Chen",
"doi-asserted-by": "publisher",
"first-page": "1560",
"issue": "6",
"journal-title": "Cell",
"key": "D4CB00230J/cit20/1",
"volume": "182",
"year": "2020"
},
{
"DOI": "10.1016/j.antiviral.2021.105033",
"author": "Martin",
"doi-asserted-by": "publisher",
"first-page": "105033",
"journal-title": "Antiviral Res.",
"key": "D4CB00230J/cit21/1",
"volume": "188",
"year": "2021"
},
{
"DOI": "10.1128/AAC.00957-12",
"author": "Adedeji",
"doi-asserted-by": "publisher",
"first-page": "4718",
"issue": "9",
"journal-title": "Antimicrob. Agents Chemother.",
"key": "D4CB00230J/cit22/1",
"volume": "56",
"year": "2012"
},
{
"DOI": "10.1016/J.BMCL.2012.04.081",
"author": "Yu",
"doi-asserted-by": "publisher",
"first-page": "4049",
"issue": "12",
"journal-title": "Bioorg. Med. Chem. Lett.",
"key": "D4CB00230J/cit23/1",
"volume": "22",
"year": "2012"
},
{
"DOI": "10.1038/s41467-020-19770-1",
"author": "Yan",
"doi-asserted-by": "publisher",
"first-page": "1",
"issue": "1",
"journal-title": "Nat. Commun.",
"key": "D4CB00230J/cit24/1",
"volume": "11",
"year": "2020"
},
{
"DOI": "10.1093/nar/gkz409",
"author": "Jia",
"doi-asserted-by": "publisher",
"first-page": "6538",
"issue": "12",
"journal-title": "Nucleic Acids Res.",
"key": "D4CB00230J/cit25/1",
"volume": "47",
"year": "2019"
},
{
"DOI": "10.4161/RNA.27446",
"author": "Zander",
"doi-asserted-by": "publisher",
"first-page": "45",
"issue": "1",
"journal-title": "RNA Biol.",
"key": "D4CB00230J/cit26/1",
"volume": "11",
"year": "2014"
},
{
"DOI": "10.1093/nar/gkad660",
"author": "Marx",
"doi-asserted-by": "publisher",
"first-page": "9266",
"journal-title": "Nucleic Acids Res.",
"key": "D4CB00230J/cit27/1",
"volume": "17",
"year": "2023"
},
{
"DOI": "10.1016/j.bbrep.2018.10.011",
"author": "Jakob",
"doi-asserted-by": "publisher",
"first-page": "1",
"journal-title": "Biochem. Biophys. Rep.",
"key": "D4CB00230J/cit28/1",
"volume": "17",
"year": "2019"
},
{
"DOI": "10.1021/acsinfecdis.9b00220",
"author": "Ablenas",
"doi-asserted-by": "publisher",
"first-page": "2118",
"issue": "12",
"journal-title": "ACS Infect. Dis.",
"key": "D4CB00230J/cit29/1",
"volume": "5",
"year": "2019"
},
{
"DOI": "10.1021/ja027007w",
"author": "Chin",
"doi-asserted-by": "publisher",
"first-page": "9026",
"issue": "31",
"journal-title": "J. Am. Chem. Soc.",
"key": "D4CB00230J/cit30/1",
"volume": "124",
"year": "2002"
},
{
"DOI": "10.1021/cr400355w",
"author": "Lang",
"doi-asserted-by": "publisher",
"first-page": "4764",
"issue": "9",
"journal-title": "Chem. Rev.",
"key": "D4CB00230J/cit31/1",
"volume": "114",
"year": "2014"
},
{
"DOI": "10.1126/sciadv.abj4526",
"author": "Perez-Lemus",
"doi-asserted-by": "publisher",
"first-page": "4526",
"issue": "1",
"journal-title": "Sci. Adv.",
"key": "D4CB00230J/cit32/1",
"volume": "8",
"year": "2022"
},
{
"DOI": "10.1042/bcj20210201",
"author": "Zeng",
"doi-asserted-by": "publisher",
"first-page": "2405",
"issue": "13",
"journal-title": "Biochem. J.",
"key": "D4CB00230J/cit33/1",
"volume": "478",
"year": "2021"
},
{
"DOI": "10.1002/cbic.201402154",
"author": "Sun",
"doi-asserted-by": "publisher",
"first-page": "1721",
"issue": "12",
"journal-title": "ChemBioChem",
"key": "D4CB00230J/cit34/1",
"volume": "15",
"year": "2014"
},
{
"DOI": "10.1038/nchem.1919",
"author": "Wang",
"doi-asserted-by": "publisher",
"first-page": "393",
"issue": "5",
"journal-title": "Nat. Chem.",
"key": "D4CB00230J/cit35/1",
"volume": "6",
"year": "2014"
},
{
"DOI": "10.3389/fimmu.2022.912717",
"author": "Herbert",
"doi-asserted-by": "publisher",
"first-page": "2814",
"journal-title": "Front. Immunol.",
"key": "D4CB00230J/cit36/1",
"volume": "13",
"year": "2022"
},
{
"DOI": "10.1146/annurev-biochem-060713-035737",
"author": "Chin",
"doi-asserted-by": "publisher",
"first-page": "379",
"journal-title": "Annu. Rev. Biochem.",
"key": "D4CB00230J/cit37/1",
"volume": "83",
"year": "2014"
},
{
"DOI": "10.1016/j.chembiol.2014.09.002",
"author": "McKay",
"doi-asserted-by": "publisher",
"first-page": "1075",
"issue": "9",
"journal-title": "Chem. Biol.",
"key": "D4CB00230J/cit38/1",
"volume": "21",
"year": "2014"
},
{
"DOI": "10.1002/anie.201000401",
"author": "Hoogenboom",
"doi-asserted-by": "publisher",
"first-page": "3415",
"issue": "20",
"journal-title": "Angew. Chem., Int. Ed.",
"key": "D4CB00230J/cit39/1",
"volume": "49",
"year": "2010"
},
{
"DOI": "10.1039/D1SC02775A",
"author": "Berta",
"doi-asserted-by": "publisher",
"first-page": "13492",
"issue": "40",
"journal-title": "Chem. Sci.",
"key": "D4CB00230J/cit40/1",
"volume": "12",
"year": "2021"
},
{
"DOI": "10.1016/s0959-440x(00)00204-9",
"author": "Ha",
"doi-asserted-by": "publisher",
"first-page": "287",
"issue": "3",
"journal-title": "Curr. Opin. Struct. Biol.",
"key": "D4CB00230J/cit41/1",
"volume": "11",
"year": "2001"
},
{
"DOI": "10.1021/bi800773f",
"author": "Iqbal",
"doi-asserted-by": "publisher",
"first-page": "7857",
"issue": "30",
"journal-title": "Biochemistry",
"key": "D4CB00230J/cit42/1",
"volume": "47",
"year": "2008"
},
{
"DOI": "10.3390/ijms16046718",
"author": "Shrestha",
"doi-asserted-by": "publisher",
"first-page": "6718",
"issue": "4",
"journal-title": "Int. J. Mol. Sci.",
"key": "D4CB00230J/cit43/1",
"volume": "16",
"year": "2015"
},
{
"DOI": "10.1039/c3cs60201j",
"author": "Hwang",
"doi-asserted-by": "publisher",
"first-page": "1221",
"issue": "4",
"journal-title": "Chem. Soc. Rev.",
"key": "D4CB00230J/cit44/1",
"volume": "43",
"year": "2014"
},
{
"DOI": "10.1017/s1355838200000728",
"author": "Seybert",
"doi-asserted-by": "publisher",
"first-page": "1056",
"issue": "7",
"journal-title": "RNA",
"key": "D4CB00230J/cit45/1",
"volume": "6",
"year": "2000"
},
{
"DOI": "10.1007/s12154-013-0094-5",
"author": "Toseland",
"doi-asserted-by": "publisher",
"first-page": "85",
"issue": "3",
"journal-title": "J. Chem. Biol.",
"key": "D4CB00230J/cit46/1",
"volume": "6",
"year": "2013"
},
{
"DOI": "10.1016/j.tibtech.2005.10.005",
"author": "Chudakov",
"doi-asserted-by": "publisher",
"first-page": "605",
"issue": "12",
"journal-title": "Trends Biotechnol.",
"key": "D4CB00230J/cit47/1",
"volume": "23",
"year": "2005"
},
{
"DOI": "10.1021/ja027007w",
"author": "Chin",
"doi-asserted-by": "publisher",
"first-page": "9026",
"issue": "31",
"journal-title": "J. Am. Chem. Soc.",
"key": "D4CB00230J/cit48/1",
"volume": "124",
"year": "2002"
},
{
"DOI": "10.1038/nmeth.1208",
"author": "Roy",
"doi-asserted-by": "publisher",
"first-page": "507",
"issue": "6",
"journal-title": "Nat. Methods",
"key": "D4CB00230J/cit49/1",
"volume": "5",
"year": "2008"
}
],
"reference-count": 49,
"references-count": 49,
"relation": {},
"resource": {
"primary": {
"URL": "https://xlink.rsc.org/?DOI=D4CB00230J"
}
},
"score": 1,
"short-title": [],
"source": "Crossref",
"subject": [],
"subtitle": [],
"title": "Monitoring SARS-CoV-2 Nsp13 helicase binding activity using expanded genetic code techniques",
"type": "journal-article",
"update-policy": "https://doi.org/10.1039/rsc_crossmark_policy"
}
