Systematic Comparison of Beetle Luciferase-Luciferin Pairs as Sources of Near-Infrared Light for In Vitro and In Vivo Applications
Luciferases catalyze light-emitting reactions that produce a rainbow of colors from their substrates (luciferins), molecular oxygen, and often additional cofactors. These bioluminescence (BL) systems have afforded an incredible variety of basic research and medical applications. Driven by the import...
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| Format: | Article |
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MDPI AG
2022-02-01
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| Series: | International Journal of Molecular Sciences |
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| Online Access: | https://www.mdpi.com/1422-0067/23/5/2451 |
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| author | Bruce R. Branchini Danielle M. Fontaine Dawn Kohrt Brian P. Huta Allison R. Racela Benjamin R. Fort Tara L. Southworth Aldo Roda |
| author_facet | Bruce R. Branchini Danielle M. Fontaine Dawn Kohrt Brian P. Huta Allison R. Racela Benjamin R. Fort Tara L. Southworth Aldo Roda |
| author_sort | Bruce R. Branchini |
| collection | DOAJ |
| description | Luciferases catalyze light-emitting reactions that produce a rainbow of colors from their substrates (luciferins), molecular oxygen, and often additional cofactors. These bioluminescence (BL) systems have afforded an incredible variety of basic research and medical applications. Driven by the importance of BL-based non-invasive animal imaging (BLI) applications, especially in support of cancer research, new BL systems have been developed by engineering beetle luciferase (Luc) variants and synthetic substrate combinations to produce red to near-infrared (nIR) light to improve imaging sensitivity and resolution. To stimulate the application of BLI research and advance the development of improved reagents for BLI, we undertook a systematic comparison of the spectroscopic and BL properties of seven beetle Lucs with LH<sub>2</sub> and nine substrates, which included two new quinoline ring-containing analogs. The results of these experiments with purified Luc enzymes in vitro and in live HEK293T cells transfected with <i>luc</i> genes have enabled us to identify Luc/analog combinations with improved properties compared to those previously reported and to provide live cell BL data that may be relevant to in vivo imaging applications. Additionally, we found strong candidate enzyme/substrate pairs for in vitro biomarker applications requiring nIR sources with minimal visible light components. Notably, one of our new substrates paired with a previously developed Luc variant was demonstrated to be an excellent in vitro source of nIR and a potentially useful BL system for improved resolution in BLI. |
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| institution | Directory Open Access Journal |
| issn | 1661-6596 1422-0067 |
| language | English |
| last_indexed | 2024-03-09T20:39:24Z |
| publishDate | 2022-02-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | International Journal of Molecular Sciences |
| spelling | doaj.art-eacff8a83fa3445bb4f3a5a297cfe3fe2023-11-23T23:04:19ZengMDPI AGInternational Journal of Molecular Sciences1661-65961422-00672022-02-01235245110.3390/ijms23052451Systematic Comparison of Beetle Luciferase-Luciferin Pairs as Sources of Near-Infrared Light for In Vitro and In Vivo ApplicationsBruce R. Branchini0Danielle M. Fontaine1Dawn Kohrt2Brian P. Huta3Allison R. Racela4Benjamin R. Fort5Tara L. Southworth6Aldo Roda7Department of Chemistry, Connecticut College, New London, CT 06320, USADepartment of Chemistry, Connecticut College, New London, CT 06320, USADepartment of Chemistry, Connecticut College, New London, CT 06320, USADepartment of Chemistry, Connecticut College, New London, CT 06320, USADepartment of Chemistry, Connecticut College, New London, CT 06320, USADepartment of Chemistry, Connecticut College, New London, CT 06320, USADepartment of Chemistry, Connecticut College, New London, CT 06320, USADepartment of Chemistry “G. Ciamician”, University of Bologna, 40126 Bologna, ItalyLuciferases catalyze light-emitting reactions that produce a rainbow of colors from their substrates (luciferins), molecular oxygen, and often additional cofactors. These bioluminescence (BL) systems have afforded an incredible variety of basic research and medical applications. Driven by the importance of BL-based non-invasive animal imaging (BLI) applications, especially in support of cancer research, new BL systems have been developed by engineering beetle luciferase (Luc) variants and synthetic substrate combinations to produce red to near-infrared (nIR) light to improve imaging sensitivity and resolution. To stimulate the application of BLI research and advance the development of improved reagents for BLI, we undertook a systematic comparison of the spectroscopic and BL properties of seven beetle Lucs with LH<sub>2</sub> and nine substrates, which included two new quinoline ring-containing analogs. The results of these experiments with purified Luc enzymes in vitro and in live HEK293T cells transfected with <i>luc</i> genes have enabled us to identify Luc/analog combinations with improved properties compared to those previously reported and to provide live cell BL data that may be relevant to in vivo imaging applications. Additionally, we found strong candidate enzyme/substrate pairs for in vitro biomarker applications requiring nIR sources with minimal visible light components. Notably, one of our new substrates paired with a previously developed Luc variant was demonstrated to be an excellent in vitro source of nIR and a potentially useful BL system for improved resolution in BLI.https://www.mdpi.com/1422-0067/23/5/2451bioluminescencefireflynear-infraredHEK293Tbiomarkerimaging |
| spellingShingle | Bruce R. Branchini Danielle M. Fontaine Dawn Kohrt Brian P. Huta Allison R. Racela Benjamin R. Fort Tara L. Southworth Aldo Roda Systematic Comparison of Beetle Luciferase-Luciferin Pairs as Sources of Near-Infrared Light for In Vitro and In Vivo Applications International Journal of Molecular Sciences bioluminescence firefly near-infrared HEK293T biomarker imaging |
| title | Systematic Comparison of Beetle Luciferase-Luciferin Pairs as Sources of Near-Infrared Light for In Vitro and In Vivo Applications |
| title_full | Systematic Comparison of Beetle Luciferase-Luciferin Pairs as Sources of Near-Infrared Light for In Vitro and In Vivo Applications |
| title_fullStr | Systematic Comparison of Beetle Luciferase-Luciferin Pairs as Sources of Near-Infrared Light for In Vitro and In Vivo Applications |
| title_full_unstemmed | Systematic Comparison of Beetle Luciferase-Luciferin Pairs as Sources of Near-Infrared Light for In Vitro and In Vivo Applications |
| title_short | Systematic Comparison of Beetle Luciferase-Luciferin Pairs as Sources of Near-Infrared Light for In Vitro and In Vivo Applications |
| title_sort | systematic comparison of beetle luciferase luciferin pairs as sources of near infrared light for in vitro and in vivo applications |
| topic | bioluminescence firefly near-infrared HEK293T biomarker imaging |
| url | https://www.mdpi.com/1422-0067/23/5/2451 |
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