FRET-Based Identification of mRNAs Undergoing Translation

We present proof-of-concept in vitro results demonstrating the feasibility of using single molecule fluorescence resonance energy transfer (smFRET) measurements to distinguish, in real time, between individual ribosomes programmed with several different, short mRNAs. For these measurements we use either the FRET signal generated between two tRNAs labeled with different fluorophores bound simultaneously in adjacent sites to the ribosome (tRNA-tRNA FRET) or the FRET signal generated between a labeled tRNA bound to the ribosome and a fluorescent derivative of ribosomal protein L1 (L1-tRNA FRET). With either technique, criteria were developed to identify the mRNAs, taking into account the relative activity of the mRNAs. These criteria enabled identification of the mRNA being translated by a given ribosome to within 95% confidence intervals based on the number of identified FRET traces. To upgrade the approach for natural mRNAs or more complex mixtures, the stoichiometry of labeling should be enhanced and photobleaching reduced. The potential for porting these methods into living cells is discussed

Repairing the Sickle Cell mutation. II. Effect of psoralen linker length on specificity of formation and yield of third strand-directed photoproducts with the mutant target sequence

Three identical deoxyoligonucleotide third strands with a 3′-terminal psoralen moiety attached by linkers that differ in length (N = 16, 6 and 4 atoms) and structure were examined for their ability to form triplex-directed psoralen photoproducts with both the mutant T residue of the Sickle Cell β-globin gene and the comparable wild-type sequence in linear duplex targets. Specificity and yield of UVA (365 nm) and visible (419 nm) light-induced photoadducts were studied. The total photoproduct yield varies with the linker and includes both monoadducts and crosslinks at various available pyrimidine sites. The specificity of photoadduct formation at the desired mutant T residue site was greatly improved by shortening the psoralen linker. In particular, using the N-4 linker, psoralen interaction with the residues of the non-coding duplex strand was essentially eliminated, while modification of the Sickle Cell mutant T residue was maximized. At the same time, the proportion of crosslink formation at the mutant T residue upon UV irradiation was much greater for the N-4 linker. The photoproducts formed with the wild-type target were fully consistent with its single base pair difference. The third strand with the N-4 linker was also shown to bind to a supercoiled plasmid containing the Sickle Cell mutation site, giving photoproduct yields comparable with those observed in the linear mutant target

Parameter values for Eq. 1 (Fig. 4).

a<p>X, Y refer to specific mRNAs (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038344#pone-0038344-t001" target="_blank">Table 1</a>).</p>b<p>R is the ratio of the apparent synthetic activity of mRNA-X relative to mRNA-Y in the mixture. Deviations of R from 1.0 may reflect differences in the efficiencies of initiation complex formation and of polypeptide elongation, the higher probability of detecting longer mRNAs vs. shorter mRNAs, and other factors not yet identified.</p

Multiple FV and VF events during translation of mRNA-1 and mRNA-2.

<p>(A) and (B), translation of mRNA-1, as detected by tRNA-tRNA FRET and by L1-tRNA FRET, respectively. (C) and (D), translation of mRNA-2, as detected by tRNA-tRNA FRET and by L1-tRNA FRET, respectively. Color coding: (A) and (C), as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038344#pone-0038344-g001" target="_blank">Fig. 1</a>; (B) and (D), as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038344#pone-0038344-g002" target="_blank">Fig. 2</a>.</p

mRNAs used in this study.

<p>The bold sequence for each mRNA is the translation start site, initiator fMet. Sequences are listed from 5′ to 3′ ends. The biotin tag for tethering to the microscope slide surface was at the 3′ end for mRNAs -1, -2, -5, and -6, and at the 5′ end for mRNAs -3 and -4.</p

Single FV and VF events detected by FRET between Cy3-F and Cy5-V during translation of mRNA-1 (FV: A, B) and mRNA-2 (VF: C, D).

<p>In the cartoons (A, C), fluorescent labels are shown as filled colored circles: Cy3 is green, Cy5 is red. Black and blue letters represent the tRNAs and codon triplets, respectively, for a given amino acid, using the standard single-letter abbreviations. For ease of presentation, all three tRNA sites are shown to be occupied during the entire event, but this need not be the case <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038344#pone.0038344-Chen2" target="_blank">[13]</a>. The excitation wavelength was alternated (ALEX) between 532 nm and 640 nm every other image. The traces in (B, D) show Cy3 fluorescence (green trace, 585 nm detection) and sensitized emission of Cy5 (FRET, blue trace, 680 nm detection), both under 532 nm excitation, and Cy5 fluorescence (red ALEX trace, 680 nm detection) under direct 640 nm excitation.</p

Analysis of mixtures of two input mRNAs.

<p>In all three panels, the proportion of one identified translated mRNA (Trace ID) is plotted (filled symbols) against the proportion of that mRNA in the reaction mixture (input proportion). The solid line through these points is fitted from Eq. 1 in the text, adjusting the ratio (R) of efficiencies of translation of the two mRNAs. The open symbols show corresponding corrected effective input proportions from Eq. 2 using the values for R from the fitted Eq. 1. tRNA-tRNA FRET (squares); L1-tRNA FRET (circles). The dashed straight lines are fitted to the corrected input proportions. mRNA mixtures: (A) 1 and 2; (B) 3 and 5; (C) 3 and 4. Uncertainties are 95% confidence intervals <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038344#pone.0038344-Clopper1" target="_blank">[37]</a>.</p

Single FV and VF events detected by FRET between L1^Cy3 and either Cy5.5-F or Cy5-V during translation of mRNA-1 (FV: A, B) and mRNA-2 (VF: C, D).

<p>The cartoons (A, C) are presented as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038344#pone-0038344-g001" target="_blank">Fig. 1</a>, with the addition that Cy5.5 is black. The traces (B, D) show Cy3 fluorescence (green) and sensitized emission of Cy5 (FRET, red) and Cy5.5 (FRET, black), all under 532 nm excitation. The emission filter wavelengths are listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038344#s4" target="_blank">Methods</a>. In B, The proximity of Cy5.5 to Cy3 causes an increase in intensity in the Cy5.5 sensitized emission channel, with some cross-talk into the Cy5 channel, followed by release of the Cy5.5-F and closer approach of the Cy5 dye to Cy3, followed by release of Cy5. In D, the order is reversed.</p