Analysis of RNA base modification and structural rearrangement by single-molecule real-time detection of reverse transcription - PubMed (original) (raw)

Analysis of RNA base modification and structural rearrangement by single-molecule real-time detection of reverse transcription

Igor D Vilfan et al. J Nanobiotechnology. 2013.

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.

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Figures

Figure 1

Figure 1

Single-molecule, real-time (SMRT) reverse transcription in zero-mode waveguides (ZMWs). (a) Schematic of the SMRT reverse transcription process. The RNA template (purple) is hybridized to a biotinylated DNA primer (orange) and immobilized at the ZMW bottom (I). Reverse transcriptase (RT, gray) binds to the immobilized hybrid (II). Upon initiation of reverse transcription, a correctly base-paired phospholinked nucleotide binds in the enzyme’s active site (III). The bound nucleotide can either dissociate from the complex (reverse reaction), or become incorporated into the growing DNA chain, accompanied by the release of the labeled pyrophosphate (IV). HIV RT then translocates to the next position and the reaction cycle (II) through (IV) repeats. (b) Hybrid for demonstrating SMRT reverse transcription, showing the RNA template (purple), DNA primer (orange), and resulting DNA (base-specific color-coding). (c) Example trace of SMRT reverse transcription. Fluorescence pulses are color-coded as in (b). Each block of pulses belonging to the same nucleotide is indicated above the trace. The gray block at the end of the trace likely corresponds to non-templated cognate sampling. (d-e) Magnified views of the beginning and end of the trace, respectively.

Figure 2

Figure 2

Detection of modified RNA bases through the kinetics of SMRT reverse transcription. (a-b) Sequences and example traces of a SMRT reverse transcription trace on m6A-containing and unmodified A-containing control template, respectively. Enlarged trace regions show reverse transcription signal for the underlined sequences. (c-d) Distributions of (c) interpulse durations (IPDs) and (d) pulse widths (PWs) for phospholinked T incorporations across m6A (orange) and A (blue). The insets in (c) and (d) show histograms of the experimental counts used to determine the cumulative distributions. Phospholinked T binding (k b) and dissociation (k d) rates were obtained from single exponential fits to the distributions.

Figure 3

Figure 3

Detection of m 6 A in native mRNA. (a) The section of the native mRNA containing the m6A modification sequence is shown in black with the potential site of methylation at position 905 shown in red and unmodified A at position 903 shown in dark yellow. The methyltransferase recognition consensus sequence is indicated in brown where R represents a purine, and H is a non-guanine base. (b) The main plot shows IPD distribution for phospholinked T incorporations across position 905 in native mRNA (red) and the in vitro transcribed control (blue), the Inset shows IPD distributions obtained at sequence position 903 in native mRNA (dark yellow) and the in vitro transcribed control (blue). (c) Pulse width (PW) distributions for phospholinked T incorporations across from the consensus site for m6A modification (position 905; data obtained with native mRNA and in vitro transcribed control shown in red and blue, respectively). Inset: PW distribution for phospholinked T incorporations at the unmodified adenosine control at position 903 (data obtained with native mRNA and in vitro transcribed control shown in dark yellow and blue, respectively).

Figure 4

Figure 4

Detection of RNA structure rearrangement during SMRT reverse transcription. (a) Histogram of reverse transcript lengths on E. coli 16S rRNA. The top axis shows 16S rRNA sequence position as defined by EcoCyc [17]. The left inset shows a schematic of 16S rRNA secondary structure. A section of 16S rRNA (gray rectangle) is expanded in the right inset, showing the start of SMRT reverse transcription and the 5’-end of 16S rRNA. Reverse transcribed bases are shown as solid blue circles and bases involved in secondary structures in transparent blue circles. Peaks in the histogram and their corresponding 16S rRNA sequence positions are indicated by arrows. (b) Histogram of reverse transcript lengths on human ribosomal protein S17 mRNA. Inset: Pausing probabilities along the mRNA template (blue bars), compared to calculated base-pairing probabilities of this mRNA template using models that do not allow (red) or do allow (black) for refolding of the RNA template remaining at each position in the DNA synthesis (Kinetic Trap Model and Equilibrium Model, respectively). A pause along mRNA template during SMRT reverse transcription was defined as stalling of reverse transcription for longer than 5 min (Methods).

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