A Novel Thermostable and Processive Reverse Transcriptase from a Group II Intron of <i>Anoxybacillus flavithermus</i>
Reverse transcriptases (RTs) are a family of enzymes that synthesize DNA using an RNA template and are involved in retrovirus propagation and telomere lengthening. In vitro, RTs are widely applied in various methods, including RNA-seq, RT-PCR, and RT-LAMP. Thermostable RTs from bacterial group II in...
| Main Authors: | , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
MDPI AG
2023-12-01
|
| Series: | Biomolecules |
| Subjects: | |
| Online Access: | https://www.mdpi.com/2218-273X/14/1/49 |
| _version_ | 1797344531031523328 |
|---|---|
| author | Igor P. Oscorbin Maxim L. Filipenko |
| author_facet | Igor P. Oscorbin Maxim L. Filipenko |
| author_sort | Igor P. Oscorbin |
| collection | DOAJ |
| description | Reverse transcriptases (RTs) are a family of enzymes that synthesize DNA using an RNA template and are involved in retrovirus propagation and telomere lengthening. In vitro, RTs are widely applied in various methods, including RNA-seq, RT-PCR, and RT-LAMP. Thermostable RTs from bacterial group II introns are promising tools for biotechnology due to their higher thermostability, fidelity, and processivity compared to commonly used M-MuLV RT and its mutants. However, the diversity of group II intron-encoded RTs is still understudied. In this work, we biochemically characterized a novel RT from a thermophilic bacterium, <i>Anoxybacillus flavithermus</i>, which was isolated from a hot spring in New Zealand and has an optimal growth temperature of around 60 °C. The cloned RT, named Afl RT, retained approximately 40% of the specific activity after a 45 min incubation at 50 °C. The optimal pH was 8.5, the optimal temperature was between 45 and 50 °C, and Mn<sup>2+</sup> ions were found to be an optimal cofactor. The processivity analysis with MS2 phage gRNA (3569 b) demonstrated that Afl RT elongated fully up to 36% of the template molecules. In reverse transcription and RT-qLAMP, the enzyme allowed up to 10 copies of MS2 phage genomic RNA to be detected per reaction. Thus, Afl RT holds great potential for a variety of practical applications that require the use of thermostable and processive RTs. |
| first_indexed | 2024-03-08T11:03:57Z |
| format | Article |
| id | doaj.art-1d63c5c8b2dc4f9c8e67ebd25db323a2 |
| institution | Directory Open Access Journal |
| issn | 2218-273X |
| language | English |
| last_indexed | 2024-03-08T11:03:57Z |
| publishDate | 2023-12-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Biomolecules |
| spelling | doaj.art-1d63c5c8b2dc4f9c8e67ebd25db323a22024-01-26T15:18:45ZengMDPI AGBiomolecules2218-273X2023-12-011414910.3390/biom14010049A Novel Thermostable and Processive Reverse Transcriptase from a Group II Intron of <i>Anoxybacillus flavithermus</i>Igor P. Oscorbin0Maxim L. Filipenko1Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences (ICBFM SB RAS), 8 Lavrentiev Avenue, 630090 Novosibirsk, RussiaInstitute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences (ICBFM SB RAS), 8 Lavrentiev Avenue, 630090 Novosibirsk, RussiaReverse transcriptases (RTs) are a family of enzymes that synthesize DNA using an RNA template and are involved in retrovirus propagation and telomere lengthening. In vitro, RTs are widely applied in various methods, including RNA-seq, RT-PCR, and RT-LAMP. Thermostable RTs from bacterial group II introns are promising tools for biotechnology due to their higher thermostability, fidelity, and processivity compared to commonly used M-MuLV RT and its mutants. However, the diversity of group II intron-encoded RTs is still understudied. In this work, we biochemically characterized a novel RT from a thermophilic bacterium, <i>Anoxybacillus flavithermus</i>, which was isolated from a hot spring in New Zealand and has an optimal growth temperature of around 60 °C. The cloned RT, named Afl RT, retained approximately 40% of the specific activity after a 45 min incubation at 50 °C. The optimal pH was 8.5, the optimal temperature was between 45 and 50 °C, and Mn<sup>2+</sup> ions were found to be an optimal cofactor. The processivity analysis with MS2 phage gRNA (3569 b) demonstrated that Afl RT elongated fully up to 36% of the template molecules. In reverse transcription and RT-qLAMP, the enzyme allowed up to 10 copies of MS2 phage genomic RNA to be detected per reaction. Thus, Afl RT holds great potential for a variety of practical applications that require the use of thermostable and processive RTs.https://www.mdpi.com/2218-273X/14/1/49reverse transcriptasegroup II intron<i>Anoxybacillus flavithermus</i>thermal stabilityprocessivityreverse transcription |
| spellingShingle | Igor P. Oscorbin Maxim L. Filipenko A Novel Thermostable and Processive Reverse Transcriptase from a Group II Intron of <i>Anoxybacillus flavithermus</i> Biomolecules reverse transcriptase group II intron <i>Anoxybacillus flavithermus</i> thermal stability processivity reverse transcription |
| title | A Novel Thermostable and Processive Reverse Transcriptase from a Group II Intron of <i>Anoxybacillus flavithermus</i> |
| title_full | A Novel Thermostable and Processive Reverse Transcriptase from a Group II Intron of <i>Anoxybacillus flavithermus</i> |
| title_fullStr | A Novel Thermostable and Processive Reverse Transcriptase from a Group II Intron of <i>Anoxybacillus flavithermus</i> |
| title_full_unstemmed | A Novel Thermostable and Processive Reverse Transcriptase from a Group II Intron of <i>Anoxybacillus flavithermus</i> |
| title_short | A Novel Thermostable and Processive Reverse Transcriptase from a Group II Intron of <i>Anoxybacillus flavithermus</i> |
| title_sort | novel thermostable and processive reverse transcriptase from a group ii intron of i anoxybacillus flavithermus i |
| topic | reverse transcriptase group II intron <i>Anoxybacillus flavithermus</i> thermal stability processivity reverse transcription |
| url | https://www.mdpi.com/2218-273X/14/1/49 |
| work_keys_str_mv | AT igorposcorbin anovelthermostableandprocessivereversetranscriptasefromagroupiiintronofianoxybacillusflavithermusi AT maximlfilipenko anovelthermostableandprocessivereversetranscriptasefromagroupiiintronofianoxybacillusflavithermusi AT igorposcorbin novelthermostableandprocessivereversetranscriptasefromagroupiiintronofianoxybacillusflavithermusi AT maximlfilipenko novelthermostableandprocessivereversetranscriptasefromagroupiiintronofianoxybacillusflavithermusi |