Genetic encoding of a highly photostable, long lifetime fluorescent amino acid for imaging in mammalian cells.

Acridonylalanine (Acd) is a fluorescent amino acid that is highly photostable, with a high quantum yield and long fluorescence lifetime in water. These properties make it superior to existing genetically encodable fluorescent amino acids for monitoring protein interactions and conformational changes through fluorescence polarization or lifetime experiments, including fluorescence lifetime imaging microscopy (FLIM). Here, we report the genetic incorporation of Acd using engineered pyrrolysine tRNA synthetase (RS) mutants that allow for efficient Acd incorporation in both E. coli and mammalian cells. We compare protein yields and amino acid specificity for these Acd RSs to identify an optimal construct. We also demonstrate the use of Acd in FLIM, where its long lifetime provides strong contrast compared to endogenous fluorophores and engineered fluorescent proteins, which have lifetimes less than 5 ns.

Access to Faster Eukaryotic Cell Labeling with Encoded Tetrazine Amino Acids.

Labeling of biomolecules in live eukaryotic cells has been limited by low component stability and slow reaction rates. We show that genetically encoded tetrazine amino acids in proteins reach reaction rates of 8 × 104 M-1 s-1 with sTCO reagents, making them the fastest site-specific bioorthogonal labels in eukaryotic systems. We demonstrate that tetrazine amino acids are stable on proteins and are capable of quantitative labeling with sTCO reagents. The exceptionally high reaction rate of this ligation minimizes label concentration, allowing for substoichiometric in vivo eukaryotic protein labeling where the concentration of the label is less than the concentration of the protein. This approach offers unprecedented control over the composition and stability of the protein tag. We anticipate that this system will have a broad impact on labeling and imaging studies because it can be used where all generally orthogonal PylRS/tRNA pairs are employed.

Growth Hormone Deficiency Causing Micropenis: Lessons Learned From a Well-Adjusted Adult.

This report of a 46,XY patient born with a micropenis consistent with etiology from isolated congenital growth hormone deficiency is used to (1) raise the question regarding what degree testicular testosterone exposure to the central nervous system during fetal life and early infancy has on the development of male gender identity, regardless of gender of rearing; (2) suggest the obligatory nature of timely full disclosure of medical history; (3) emphasize that virtually all 46,XY infants with functional testes and a micropenis should be initially boys except some with partial androgen insensitivity syndrome; and (4) highlight the sustaining value of a positive long-term relationship with a trusted physician (R.M.B.). When this infant presented, it was commonly considered inappropriate to gender assign an infant male whose penis was so small that an adult size was expected to be inadequate, even if the karyotype was 46,XY, and testes were functional. Concomitantly, female gender assignment was considered the appropriate decision, believing that parental rearing in the assigned gender was considered the major factor determining established adult gender identity. Full disclosure of medical information was considered inappropriate. Progress in appreciating the complexities of gender identity development, which is not yet completely understood, and sexuality, coping ability, and outcome data has resulted in a change of practice in initial gender assignment. A 46,XY individual with functional testes and verified androgen responsiveness should be assigned and reared as male, regardless of penis size. Without androgen responsiveness, the multiple factors must be carefully considered and disclosed.

Site-Specific Protein Labeling with Tetrazine Amino Acids.

Genetic code expansion is commonly used to introduce bioorthogonal reactive functional groups onto proteins for labeling. In recent years, the inverse electron demand Diels-Alder reaction between tetrazines and strained trans-cyclooctenes has increased in popularity as a bioorthogonal ligation for protein labeling due to its fast reaction rate and high in vivo stability. We provide methods for the facile synthesis of a tetrazine containing amino acid, Tet-v2.0, and the site-specific incorporation of Tet-v2.0 into proteins via genetic code expansion. Furthermore, we demonstrate that proteins containing Tet-v2.0 can be quickly and efficiently reacted with strained alkene labels at low concentrations. This chemistry has enabled the labeling of protein surfaces with fluorophores, inhibitors, or common posttranslational modifications such as glycosylation or lipidation.

Improving target amino acid selectivity in a permissive aminoacyl tRNA synthetase through counter-selection.

The amino acid acridon-2-ylalanine (Acd) can be a valuable probe of protein dynamics, either alone or as part of a Förster resonance energy transfer (FRET) or photo-induced electron transfer (eT) probe pair. We have previously reported the genetic incorporation of Acd by an aminoacyl tRNA synthetase (RS). However, this RS, developed from a library of permissive RSs, also incorporates N-phenyl-aminophenylalanine (Npf), a trace byproduct of one Acd synthetic route. We have performed negative selections in the presence of Npf and analyzed the selectivity of the resulting AcdRSs by in vivo protein expression and detailed kinetic analyses of the purified RSs. We find that selection conferred a ∼50-fold increase in selectivity for Acd over Npf, eliminating incorporation of Npf contaminants, and allowing one to use a high yielding Acd synthetic route for improved overall expression of Acd-containing proteins. More generally, our report also provides a cautionary tale on the use of permissive RSs, as well as a strategy for improving selectivity for the target amino acid.

Ideal Bioorthogonal Reactions Using A Site-Specifically Encoded Tetrazine Amino Acid.

Bioorthogonal reactions for labeling biomolecules in live cells have been limited by slow reaction rates or low component selectivity and stability. Ideal bioorthogonal reactions with high reaction rates, high selectivity, and high stability would allow for stoichiometric labeling of biomolecules in minutes and eliminate the need to wash out excess labeling reagent. Currently, no general method exists for controlled stoichiometric or substoichiometric labeling of proteins in live cells. To overcome this limitation, we developed a significantly improved tetrazine-containing amino acid (Tet-v2.0) and genetically encoded Tet-v2.0 with an evolved aminoacyl-tRNA synthetase/tRNA(CUA) pair. We demonstrated in cellulo that protein containing Tet-v2.0 reacts selectively with cyclopropane-fused trans-cyclooctene (sTCO) with a bimolecular rate constant of 72,500 ± 1660 M(-1) s(-1) without reacting with other cellular components. This bioorthogonal ligation of Tet-v2.0-protein reacts in cellulo with substoichiometric amounts of sTCO-label fast enough to remove the labeling reagent from media in minutes, thereby eliminating the need to wash out label. This ideal bioorthogonal reaction will enable the monitoring of a larger window of cellular processes in real time.

1,2,4-Triazines Are Versatile Bioorthogonal Reagents.

A new class of bioorthogonal reagents, 1,2,4-triazines, is described. These scaffolds are stable in biological media and capable of robust reactivity with trans-cyclooctene (TCO). The enhanced stability of the triazine scaffold enabled its direct use in recombinant protein production. The triazine-TCO reaction can also be used in tandem with other bioorthogonal cycloaddition reactions. These features fill current voids in the bioorthogonal toolkit.

History of growth hormone therapy.

The first human to receive GH therapy was in 1956; it was of bovine origin and was given for 3 wk for metabolic balance studies revealing no effects. By 1958, three separate laboratories utilizing different extraction methods retrieved hGH from human pituitaries, purified it and used for clinical investigation. By 1959 presumed GHD patients were being given native hGH collected and extracted by various methods. Since 1 mg of hGH was needed to treat one patient per day, >360 human pituitaries were needed per patient per year. Thus, the availability of hGH was limited and was awarded on the basis of clinical research protocols approved by the National Pituitary Agency (NPA) established in 1961. hGH was dispensed and injected on a milligram weight basis with varied concentrations between batches from 0.5 units/mg to 2.0 units/mg of hGH. By 1977 a centralized laboratory was established to extract all human pituitaries in the US, this markedly improved the yield of hGH obtained and most remarkably, hGH of this laboratory was never associated with Creutzfeld-Jacob disease (CJD) resulting from the injection of apparently prior- contaminated hGH produced years earlier. However, widespread rhGH use was not possible even if a pituitary from each autopsy performed in the US was collected, this would only permit therapy for about 4,000 patients. Thus, the mass production of rhGH required the identification of the gene structure of the hormone, methodology that began in 1976 to make insulin by recombinant technology. Serendipity was manifest in 1985 when patients who had received hGH years previously were reported to have died of CJD. This led to the discontinuation of the distribution and use of hGH, at a time when a synthetic rhGH became available for clinical use. The creation of a synthetic rhGH was accompanied by unlimited supplies of hGH for investigation and therapy. However, the appropriate use and the potential abuse of this hormone are to be dealt with. The illegitimate use of rhGH, unequivocally the abuse by athletes is, and should be, of primary concern to society and should be halted. The abuse of prescribing rhGH in an attempt to retard the aging process also should receive attention.

Longitudinal assessment of hormonal and physical alterations during normal puberty in boys. IV: Predictions of adult height by the Bayley-Pinneau, Roche-Wainer-Thissen, and Tanner-Whitehouse methods compared.

To determine how accurately the Roche-Wainer-Thissen (RWT), Tanner-Whitehouse (TW2), and Bayley-Pinneau (BP) prediction models estimated adult height, serial height predictions were made for 23 healthy boys (mean initial age 10.4 ± 1.1 years) every 8 months from 8-15 years of age. The RWT model was tested using Greulich-Pyle (RWT-GP) and Fels (RWT-Fels) bone ages. Stature was measured every 4 months until near final height was attained (growth rate <1cm · 8 mo-1). Mean age at near final height was 18.4 ± 1.4 years. To assure that the predictions were as accurate and precise as possible, bone age assessments were made by experts in each method. To investigate the influence of maturation on the predictions, the boys were grouped by Fels bone ages: <11 yr, 11-13.99 yr, and 14-14.99 yr. Comparison of the prediction bias and of the root mean square errors (RMSE) showed that the TW2 model gave the most accurate results, followed by the RWT and BP models. The adult height was generally underpredicted by the TW2 model and overpredicted by the RWT and BP models. The RMSE was reduced for each of the models as the bone age approached maturity. The TW2 model had the smallest average RMSE in all bone age groups. In the <11 yr bone age group, the RWT-Fels, RWT-GP, and BP models produced RMSEs that were 16.4%, 18.4%, 62.1%, respectively, greater than the TW2 model. For the 11-13.99 yr group, RMSE by the RWT-Fels, RWT-GP, and BP models were 7.5%, 18.0%, and 15.2%, respectively, greater than the TW2 model. In the 14-14.99 yr group the RWT-GP model had a 45.5% greater RMSE than the TW2 model, whereas the RWT-Fels model produced a RMSE only 15.2% greater than TW2. The RWT-Fels model produced a lower RMSE than the RWT-GP model for all bone age groups. Although the data are probably as accurate and precise as presently possible, biologically significant error remains, especially with overprediction of adult height in normally growing boys by the BP and RWT models. It is recommended that regardless of the prediction model implemented, caution be used when advising patients of their predicted adult height since all of the models tested had outlying predictions. Am. J. Hum. Biol. 9:371-380, 1997. © 1997 Wiley-Liss, Inc.