Naturally occurring cellular RNAs contain an impressive number of chemically distinct modified residues which appear posttranscriptionally, as a result of specific action of the corresponding RNA modification enzymes. Over 100 different chemical modifications have been identified and characterized up to now. Identification of the chemical nature and exact position of these modifications is typically based on 2D-TLC analysis of nucleotide digests, on HPLC coupled with mass spectrometry, or on the use of primer extension by reverse transcriptase. However, many modified nucleotides are silent in reverse transcription, since the presence of additional chemical groups frequently does not change base-pairing properties. In this paper, we give a summary of various chemical approaches exploiting the specific reactivity of modified nucleotides in RNA for their detection. 1. Introduction Native cellular RNAs contain numerous modified residues resulting from specific action of various RNA modification enzymes. These RNA modifications are ubiquitous in nature, but the specific modification profile varies depending on the organism. Over 100 chemically distinct modified nucleotides have been identified so far mostly in tRNAs, rRNAs, snRNAs and some snoRNAs. From the chemical point of view, these modifications are highly diverse and almost any position of the nucleobases as well as the 2′-OH of the ribose has been found to be a target of modification enzymes (see Table 1 and below) [1–3]. Table 1: Known RNA modifications and their abbreviations and symbols (modified nucleosides mentioned in the text are indicated in bold). Identification of the chemical nature and localization of the modified nucleotides even in highly abundant RNAs represents a laborious and time-consuming task. Moreover, the analysis of low abundant cellular RNAs is extremely difficult due to limited access to highly purified RNA species required for most types of analysis, like HPLC or mass spectrometry [4–7]. One alternative to this consists in direct analysis of underrepresented RNA species in total cellular RNA by reverse transcription (RT) using specific DNA primers [8]. This generally allows the sequencing of a given RNA, but the information on its modified nucleotide content is still missing. The use of specific chemical reagents reviewed in this survey explores the particular reactivity of a given modified residue and may considerably help in the interpretation of an RT profile. Another area for the use of specific chemical reactions is RNA analysis and sequencing by various types of mass
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