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

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.

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

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

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

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

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

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.