Rapid and sensitive detection of native glycoRNAs
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Abstract
Chemical tools enable precise characterization of many biopolymers, including glycoconjugates. Metabolic chemical reporters enabled the discovery of glycoRNAs, however they have certain limitations due the requirement of having living cells to incorporate the modified sugar. Here we develop a periodate oxidation and aldehyde ligation method to detect and characterize native sialoglycoRNAs, termed rPAL. With optimized RNA biochemistry to enhance recovery and analysis of small RNAs, we show rPAL is at least an order of magnitude more sensitive than previous methods for detecting sialoglycoRNAs. These improvements allow rPAL to detect sialoglycoRNA from human clinical samples as demonstrated by defining the abundance and patterns of sialoglycoRNAs from sorted populations of peripheral blood mononuclear cells. The sensitivity, robustness, and flexibility of rPAL will allow greater access towards characterizing glycoRNA biology.
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Finally, to establish the high MW signal is indeed sialoglycoRNA, we evaluated its sensitivity to enzymatic digestion. Incubation of purified RNA with RNase or sialidase and subsequent rPAL-labeling results in total loss of all rPAL signal after RNase but selective and complete loss of the high MW signal after sialidase treatment (Figure 1E), consistent with this rPAL-based method labeling sialic acids in sialoglycoRNAs.
Great and convincing specificity control. This would be a better way of confirming Neu5Ac-specific labeling than my suggestion above because it can be used in a culture-independent fashion.
This is an archived comment originally written by Peter Thuy-Boun
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The ability to label fractionally more of the sialoglycoRNAs in the cell opened the possibility that we may be detecting novel sialoglycoRNAs in addition to the forms found initially with Ac4ManNAz. For example, while we noticed clear and significant loss of rPAL signal after NGI-1 and Kif treatment of HeLa cells, the effects were less than what we had previously seen only with Ac4ManNAz-labeling
Very cool!
This is an archived comment originally written by Peter Thuy-Boun
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While rPAL improves sensitivity of apparent high molecular weight (MW) glycoRNA species, it also induces
Do you think combining Ac4ManNAz and rPAL labeling could be a good way to both specifically identify Neu5Ac-ligated RNA and amplify that signal using orthogonal labels (perhaps Biotin and a FLAG tag) with different fluorophores?
This is an archived comment originally written by Peter Thuy-Boun
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Extracting RNA from Ac4ManNAz labeled HeLa cells with TRIzol and either performing copper-free click1 or rPAL, we found that rPAL generates approximately 150x the amount of signal (Figure 1C, lanes 1-3 vs 7-9).
Would this difference in signal intensity be smaller if the copper-free click reaction were incubated longer or if a higher concentration of DBCO-biotin were used?
This is an archived comment originally written by Peter Thuy-Boun
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sialoglycoRNAs
This approach seems to work well for carbohydrates susceptible to periodate oxidation like Neu5Ac, but do you think there are strategies that could target non-NeuAc-containing RNA-glycoconjugates?
This is an archived comment originally written by Peter Thuy-Boun
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The ability to label fractionally more of the sialoglycoRNAs in the cell opened the possibility that we may be detecting novel sialoglycoRNAs in addition to the forms found initially with Ac4ManNAz. For example, while we noticed clear and significant loss of rPAL signal after NGI-1 and Kif treatment of HeLa cells, the effects were less than what we had previously seen only with Ac4ManNAz-labeling
Very cool!
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Finally, to establish the high MW signal is indeed sialoglycoRNA, we evaluated its sensitivity to enzymatic digestion. Incubation of purified RNA with RNase or sialidase and subsequent rPAL-labeling results in total loss of all rPAL signal after RNase but selective and complete loss of the high MW signal after sialidase treatment (Figure 1E), consistent with this rPAL-based method labeling sialic acids in sialoglycoRNAs.
Great and convincing specificity control. This would be a better way of confirming Neu5Ac-specific labeling than my suggestion above because it can be used in a culture-independent fashion.
-
Extracting RNA from Ac4ManNAz labeled HeLa cells with TRIzol and either performing copper-free click1 or rPAL, we found that rPAL generates approximately 150x the amount of signal (Figure 1C, lanes 1-3 vs 7-9).
Would this difference in signal intensity be smaller if the copper-free click reaction were incubated longer or if a higher concentration of DBCO-biotin were used?
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While rPAL improves sensitivity of apparent high molecular weight (MW) glycoRNA species, it also induces background labeling; most notably the 18S rRNA and the small RNA pool (Figure 1C and elsewhere).
Do you think combining Ac4ManNAz and rPAL labeling could be a good way to both specifically identify Neu5Ac-ligated RNA and amplify that signal using orthogonal labels (perhaps Biotin and a FLAG tag) with different fluorophores?
-
sialoglycoRNA
This approach seems to work well for carbohydrates susceptible to periodate oxidation like Neu5Ac, but do you think there are strategies that could target non-NeuAc-containing RNA-glycoconjugates?
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