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. 2022 Sep 3;12(9):1233.
doi: 10.3390/biom12091233.

t6A and ms2t6A Modified Nucleosides in Serum and Urine as Strong Candidate Biomarkers of COVID-19 Infection and Severity

Yu Nagayoshi  1   2 , Kayo Nishiguchi  1 , Ryosuke Yamamura  1   2 , Takeshi Chujo  1 , Hiroyuki Oshiumi  3 , Hiroko Nagata  1 , Hitomi Kaneko  1 , Keiichi Yamamoto  4 , Hirotomo Nakata  5 , Korin Sakakida  1   6 , Akihiro Kunisawa  7 , Masataka Adachi  2 , Yutaka Kakizoe  2 , Takanori Mizobe  8 , Jun-Ichi Kuratsu  9 , Shinya Shimada  10 , Yasushi Nakamori  11 , Masao Matsuoka  5 , Masashi Mukoyama  2 , Fan-Yan Wei  12 , Kazuhito Tomizawa  1
Affiliations

t6A and ms2t6A Modified Nucleosides in Serum and Urine as Strong Candidate Biomarkers of COVID-19 Infection and Severity

Yu Nagayoshi et al. Biomolecules. .

Abstract

SARS-CoV-2 infection alters cellular RNA content. Cellular RNAs are chemically modified and eventually degraded, depositing modified nucleosides into extracellular fluids such as serum and urine. Here we searched for COVID-19-specific changes in modified nucleoside levels contained in serum and urine of 308 COVID-19 patients using liquid chromatography-mass spectrometry (LC-MS). We found that two modified nucleosides, N6-threonylcarbamoyladenosine (t6A) and 2-methylthio-N6-threonylcarbamoyladenosine (ms2t6A), were elevated in serum and urine of COVID-19 patients. Moreover, these levels were associated with symptom severity and decreased upon recovery from COVID-19. In addition, the elevation of similarly modified nucleosides was observed regardless of COVID-19 variants. These findings illuminate specific modified RNA nucleosides in the extracellular fluids as biomarkers for COVID-19 infection and severity.

Keywords: COVID-19; LC-MS; modified nucleosides.

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Conflict of interest statement

K.T. was funded by Shimadzu Corporation and AiSTI Science Corporation. Other authors have no competing interest.

Figures

Figure 1
Figure 1
Screening of candidate modified nucleosides by SARS-CoV-2. (a) Heatmap analysis of modified nucleoside levels in extracted RNA from SARS-CoV-2-infected HEK293 cells. Color scale shows the auto-scaled relative mean of n = 3 biological replicates. ψ: pseudouridine; Cm: 2′-O-methylcytidine; m1A: N1-methyladenosine; m6A: N6-methyladenosine; Um: 2′-O-methyluridine; Gm: 2′-O-methylguanosine; Im: 2′-O-methylinosine; m22G: N2,N2-dimethylguanosine; t6A: N6-threonylcarbamoyladenosine; Am: 2′-O-methyladenosine; ms2t6A: 2-methylthio-N6-threonylcarbamoyladenosine; m6,2A: N6,N6-dimethyladenosine; m6t6A: N6-methyl-threonylcarbamoyladenosine; m6Am: N6,2′-O-dimethyladenosine; D: dihydrouridine; U: uridine; C: cytidine; G: guanosine; A: adenosine. (b) Chemical structures of t6A and ms2t6A. Modified residues are depicted in red.
Figure 2
Figure 2
SARS-CoV-2 infection causes elevation of t6A and ms2t6A levels in urine. (a,b) Measurements of t6A (a) and ms2t6A (b) in urine and comparison with healthy volunteers. LC-MS peak areas of t6A and ms2t6A divided by urine creatinine levels are shown. ** p < 0.01, *** p < 0.001 by Mann–Whitney U test. (c,d) ROC analysis for measurements of t6A (c) and ms2t6A (d) in urine was performed for calculation of sensitivity and specificity.
Figure 3
Figure 3
The comparison of t6A and ms2t6A levels in the urine of COVID-19 patients with other infectious diseases. (a,b) Comparison of t6A (a) and ms2t6A (b) levels in the urine of patients with viral infection or bacterial pneumoniae. LC-MS peak areas of t6A and ms2t6A divided by urine creatinine levels are shown. * p < 0.05, *** p < 0.001, **** p < 0.0001 by Kruskal–Wallis test and Dunn’s multiple comparison test.
Figure 4
Figure 4
Elevation of t6A and ms2t6A in serum of COVID-19 patients. (a,b) Measurements of t6A (a) and ms2t6A (b) in serum of COVID-19 patients and comparison with healthy volunteers. LC-MS peak areas of t6A or ms2t6A divided by LC-MS peak areas of adenosine are shown. **** p < 0.0001 by Mann–Whitney U test. (c,d) ROC analysis for t6A (c) and ms2t6A (d) levels in serum was performed for calculation of sensitivity and specificity.
Figure 5
Figure 5
Elevations of t6A and ms2t6A in serum of COVID-19 patients correlate with severity and recovery of COVID-19. (a,b) Elevation of t6A (a) and ms2t6A (b) in serum categorized by COVID-19 severity. LC-MS peak areas of t6A or ms2t6A divided by LC-MS peak areas of adenosine are shown. ** p < 0.01, **** p < 0.0001 by Kruskal–Wallis test and Dunn’s multiple comparison test. (c,d) Comparison of t6A (c) and ms2t6A (d) levels in serum of COVID-19 patients at the time of infection and recovery. A recovery period was defined by the resolution of fever and other symptoms. LC-MS peak areas of t6A or ms2t6A divided by LC-MS peak areas of adenosine are shown. **** p < 0.0001 by Wilcoxon rank-sum test.
Figure 6
Figure 6
Elevation of t6A and ms2t6A levels in serum of patients infected by different SARS-CoV-2 strains. (a,b) Measurements of t6A (a) and ms2t6A (b) in serum of COVID-19 patients infected with α strain or δ strain compared with healthy volunteers. LC-MS peak areas of t6A or ms2t6A divided by LC-MS peak areas of adenosine are shown. **** p < 0.0001 by Kruskal–Wallis test and Dunn’s multiple comparison test. ns, not significant.
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