title: Analysis of RNA base modification and structural rearrangement by single-molecule real-time detection of reverse transcription.
abstract: BACKGROUND
  Zero-mode waveguides (ZMWs) are photonic nanostructures that create highly confined optical observation volumes, thereby allowing single-molecule-resolved biophysical studies at relatively high concentrations of fluorescent molecules. This principle has been successfully applied in single-molecule, real-time (SMRT®) DNA sequencing for the detection of DNA sequences and DNA base modifications. In contrast, RNA sequencing methods cannot provide sequence and RNA base modifications concurrently as they rely on complementary DNA (cDNA) synthesis by reverse transcription followed by sequencing of cDNA. Thus, information on RNA modifications is lost during the process of cDNA synthesis.
  
  
  RESULTS
  Here we describe an application of SMRT technology to follow the activity of reverse transcriptase enzymes synthesizing cDNA on thousands of single RNA templates simultaneously in real time with single nucleotide turnover resolution using arrays of ZMWs. This method thereby obtains information from the RNA template directly. The analysis of the kinetics of the reverse transcriptase can be used to identify RNA base modifications, shown by example for N6-methyladenine (m6A) in oligonucleotides and in a specific mRNA extracted from total cellular mRNA. Furthermore, the real-time reverse transcriptase dynamics informs about RNA secondary structure and its rearrangements, as demonstrated on a ribosomal RNA and an mRNA template.
  
  
  CONCLUSIONS
  Our results highlight the feasibility of studying RNA modifications and RNA structural rearrangements in ZMWs in real time. In addition, they suggest that technology can be developed for direct RNA sequencing provided that the reverse transcriptase is optimized to resolve homonucleotide stretches in RNA.
authors.affiliation: Pacific Biosciences, Menlo Park, CA 94025, USA.
authors.forename: Igor D
authors.initials: ID
authors.lastname: Vilfan
authors.forename: Yu-Chih
authors.initials: YC
authors.lastname: Tsai
authors.forename: Tyson A
authors.initials: TA
authors.lastname: Clark
authors.forename: Jeffrey
authors.initials: J
authors.lastname: Wegener
authors.forename: Qing
authors.initials: Q
authors.lastname: Dai
authors.forename: Chengqi
authors.initials: C
authors.lastname: Yi
authors.forename: Tao
authors.initials: T
authors.lastname: Pan
authors.forename: Stephen W
authors.initials: SW
authors.lastname: Turner
authors.forename: Jonas
authors.initials: J
authors.lastname: Korlach
mesh_terms: D018076:DNA, Complementary / Q000032:analysis / Q000235:genetics; D015321:Gene Rearrangement; D007700:Kinetics; D049329:Nanostructures / Q000737:chemistry; D036103:Nanotechnology / Q000379:methods*; D009711:Nucleotides / Q000737:chemistry; D012333:RNA, Messenger / Q000032:analysis* / Q000737:chemistry / Q000235:genetics; D012194:RNA-Directed DNA Polymerase / Q000235:genetics / Q000378:metabolism; D048348:Reverse Transcription; D017422:Sequence Analysis, DNA / Q000379:methods
chemical_list: D018076:DNA, Complementary; D009711:Nucleotides; D012333:RNA, Messenger; D012194:RNA-Directed DNA Polymerase
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