Multicolor plate reader fluorescence calibration

This is a pre-copyedited, author-produced version of an article accepted for publication in Synthetic Biology following peer review. The version of record Jacob Beal, Cheryl A Telmer, Alejandro Vignoni, Yadira Boada, Geoff S Baldwin, Liam Hallett, Taeyang Lee, Vinoo Selvarajah, Sonja Billerbeck, Bradley Brown, Guo-nan Cai, Liang Cai, Edward Eisenstein, Daisuke Kiga, David Ross, Nina Alperovich, Noah Sprent, Jaclyn Thompson, Eric M Young, Drew Endy, Traci Haddock-Angelli, Multicolor plate reader fluorescence calibration, Synthetic Biology, Volume 7, Issue 1, 2022, ysac010, https://doi.org/10.1093/synbio/ysac010, is available online at: https://doi.org/10.1093/synbio/ysac010.[EN] Plate readers are commonly used to measure cell growth and fluorescence, yet the utility and reproducibility of plate reader data is limited by the fact that it is typically reported in arbitrary or relative units. We have previously established a robust serial dilution protocol for calibration of plate reader measurements of absorbance to estimated bacterial cell count and for green fluorescence from proteins expressed in bacterial cells to molecules of equivalent fluorescein. We now extend these protocols to calibration of red fluorescence to the sulforhodamine-101 fluorescent dye and blue fluorescence to Cascade Blue. Evaluating calibration efficacy via an interlaboratory study, we find that these calibrants do indeed provide comparable precision to the prior calibrants and that they enable effective cross-laboratory comparison of measurements of red and blue fluorescence from proteins expressed in bacterial cells.This work was supported in part by the following funding sources: J.B. was supported by Air Force Research Laboratory (AFRL) and DARPA contract FA8750-17-C-0184. N.S. was supported by funding from the BBSRC under award BB/M011178/1. G.B., L.H., and T.L. were supported by the EPSRC under award EP/R034915/1 and EP/S022856/1. G.C. and L.C. were supported by funds from YF Capital and the National Top Talent Undergraduate Training Program, China. A.V. and Y.B. were funded by Grant MINECO/AEI, EU DPI2017-82896-C2-1-R and MCIN/AEI/10.13039/501100011033 grant number PID2020117271RB-C21. Y.B. was supported by Secretaria de Educaci ' on Superior, Ciencia, Tecnologia e Innovacion-Ecuador (Scholarship Convocatoria Abierta 2011). D.K. was supported by JST, CREST Grant Number JPMJCR21N4, Japan. E.Y. was supported by the National Science Foundation under Grant No. 1939860. J.T. was supported by the Office of the Director of National Intelligence (ODNI), Intelligence Advanced Research Projects Activity (IARPA) under Finding Engineering Linked Indicators (FELIX) program contract N66001-18-C-4507. This document does not contain technology or technical data controlled under either the U.S. International Traffic in Arms Regulations or the U.S. Export Administration Regulations. Views, opinions, and/or findings expressed are those of the author(s) and should not be interpreted as representing the official views or policies of the Department of Defense or the U.S. Government.Beal, J.; Telmer, CA.; Vignoni, A.; Boada-Acosta, YF.; Baldwin, GS.; Hallett, L.; Lee, T.... (2022). Multicolor plate reader fluorescence calibration. Synthetic Biology. 7(1):1-9. https://doi.org/10.1093/synbio/ysac010197

Multicolor plate reader fluorescence calibration

Plate readers are commonly used to measure cell growth and fluorescence, yet the utility and reproducibility of plate reader data is limited by the fact that it is typically reported in arbitrary or relative units. We have previously established a robust serial dilution protocol for calibration of plate reader measurements of absorbance to estimated bacterial cell count and for green fluorescence from proteins expressed in bacterial cells to molecules of equivalent fluorescein. We now extend these protocols to calibration of red fluorescence to the sulforhodamine-101 fluorescent dye and blue fluorescence to Cascade Blue. Evaluating calibration efficacy via an interlaboratory study, we find that these calibrants do indeed provide comparable precision to the prior calibrants and that they enable effective cross-laboratory comparison of measurements of red and blue fluorescence from proteins expressed in bacterial cells

Enhanced Hybridization Selectivity Using Structured GammaPNA Probes

High affinity nucleic acid analogues such as gammaPNA (γPNA) are capable of invading stable secondary and tertiary structures in DNA and RNA targets but are susceptible to off-target binding to mismatch-containing sequences. We introduced a hairpin secondary structure into a γPNA oligomer to enhance hybridization selectivity compared with a hairpin-free analogue. The hairpin structure features a five base PNA mask that covers the proximal five bases of the γPNA probe, leaving an additional five γPNA bases available as a toehold for target hybridization. Surface plasmon resonance experiments demonstrated that the hairpin probe exhibited slower on-rates and faster off-rates (i.e., lower affinity) compared with the linear probe but improved single mismatch discrimination by up to a factor of five, due primarily to slower on-rates for mismatch vs. perfect match targets. The ability to discriminate against single mismatches was also determined in a cell-free mRNA translation assay using a luciferase reporter gene, where the hairpin probe was two-fold more selective than the linear probe. These results validate the hairpin design and present a generalizable approach to improving hybridization selectivity

Detection and Assignment of Mutations and Minihaplotypes in Human DNA Using Peptide Mass Signature Genotyping (PMSG): Application to the Human RDS/Peripherin Gene

Peptide mass-signature genotyping (PMSG) is a scanning genotyping method that identifies mutations and polymorphisms by translating the sequence of interest in more than one reading frame and measuring the masses of the resulting peptides by mass spectrometry. PMSG was applied to the RDS/peripherin gene of 16 individuals from a family exhibiting autosomal dominant macular degeneration. The method revealed an A→T transversion in the 5′ splice site of intron 2 that is the likely cause of the disease. It also revealed four different minihaplotypes in exon 3 that represent particular combinations of SNPs at four different locations. This study demonstrates the utility of PMSG for identifying and characterizing point mutations and local minihaplotypes that are not readily analyzed by other approaches

In Vitro Reversible Translation Control Using γPNA Probes

On-demand regulation of gene expression in living cells is a central goal of chemical biology and antisense therapeutic development. While significant advances have allowed regulatory modulation through inserted genetic elements, on-demand control of the expression/translation state of a given native gene by complementary sequence interactions remains a technical challenge. Toward this objective, we demonstrate the reversible suppression of a luciferase gene in cell-free translation using Watson–Crick base pairing between the mRNA and a complementary γ-modified peptide nucleic acid (γPNA) sequence with a noncomplementary toehold. Exploiting the favorable thermodynamics of γPNA−γPNA interactions, the antisense sequence can be removed by hybridization of a second, fully complementary γPNA, through a strand displacement reaction, allowing translation to proceed. Complementary RNA is also shown to displace the bound antisense γPNA, opening up possibilities of in vivo regulation by native gene expression

RNA G‑Quadruplex Invasion and Translation Inhibition by Antisense γ‑Peptide Nucleic Acid Oligomers

We have examined the abilities of three complementary γ-peptide nucleic acid (γPNA) oligomers to invade an RNA G-quadruplex and potently inhibit translation of a luciferase reporter transcript containing the quadruplex-forming sequence (QFS) within its 5′-untranslated region. All three γPNA oligomers bind with low nanomolar affinities to an RNA oligonucleotide containing the QFS. However, while all probes inhibit translation with low to midnanomolar IC₅₀ values, the γPNA designed to hybridize to the first two G-tracts of the QFS and adjacent 5′-overhanging nucleotides was 5–6 times more potent than probes directed to either the 3′-end or internal regions of the target at 37 °C. This position-dependent effect was eliminated after the probes and target were preincubated at an elevated temperature prior to translation, demonstrating that kinetic effects exert significant control over quadruplex invasion and translation inhibition. We also found that antisense γPNAs exhibited similarly potent effects against luciferase reporter transcripts bearing QFS motifs having G₂, G₃, or G₄ tracts. Finally, our results indicate that γPNA oligomers exhibit selectivity and/or potency higher than those of other antisense molecules such as standard PNA and 2′-OMe RNA previously reported to target G-quadruplexes in RNA