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antiviral effects against sars cov 2 infection  (TargetMol)


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    TargetMol antiviral effects against sars cov 2 infection
    Schematic overview of the <t>anti-SARS-CoV-2</t> drug screening procedure. ( a ) ACE2 targeted inhibitors and compounds from the natural product library were applied prior to infection with SARS-CoV-2 (pre-infection treatment) while compounds from the FDA-approved library and the flavonoid library were applied post infection (post- infection treatment). Out of 2191 compounds tested, 121 displayed viral CPE reductions and 7 were selected for further validations using Vero E6 and HuH7 cell lines and hNEC. ( b ) Uninfected cells, infected cells treated with 0.1% DMSO, and infected cells treated with remdesivir were included as controls in the screen. Following treatment with citicoline, pravastatin sodium, tenofovir alafenamide, imatinib mesylate, calcitriol, dexlansoprazole, and prochlorperazine dimaleate, reduced CPE was observed when compared to the DMSO control.
    Antiviral Effects Against Sars Cov 2 Infection, supplied by TargetMol, used in various techniques. Bioz Stars score: 96/100, based on 118 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/replication/Anti-Viral+Compound+Library/pmc10058714-73-9-27
    Average 96 stars, based on 118 article reviews
    antiviral effects against sars cov 2 infection - by Bioz Stars, 2026-11
    96/100 stars

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    1) Product Images from "Evaluation of In Vitro and In Vivo Antiviral Activities of Vitamin D for SARS-CoV-2 and Variants"

    Article Title: Evaluation of In Vitro and In Vivo Antiviral Activities of Vitamin D for SARS-CoV-2 and Variants

    Journal: Pharmaceutics

    doi: 10.3390/pharmaceutics15030925

    Schematic overview of the anti-SARS-CoV-2 drug screening procedure. ( a ) ACE2 targeted inhibitors and compounds from the natural product library were applied prior to infection with SARS-CoV-2 (pre-infection treatment) while compounds from the FDA-approved library and the flavonoid library were applied post infection (post- infection treatment). Out of 2191 compounds tested, 121 displayed viral CPE reductions and 7 were selected for further validations using Vero E6 and HuH7 cell lines and hNEC. ( b ) Uninfected cells, infected cells treated with 0.1% DMSO, and infected cells treated with remdesivir were included as controls in the screen. Following treatment with citicoline, pravastatin sodium, tenofovir alafenamide, imatinib mesylate, calcitriol, dexlansoprazole, and prochlorperazine dimaleate, reduced CPE was observed when compared to the DMSO control.
    Figure Legend Snippet: Schematic overview of the anti-SARS-CoV-2 drug screening procedure. ( a ) ACE2 targeted inhibitors and compounds from the natural product library were applied prior to infection with SARS-CoV-2 (pre-infection treatment) while compounds from the FDA-approved library and the flavonoid library were applied post infection (post- infection treatment). Out of 2191 compounds tested, 121 displayed viral CPE reductions and 7 were selected for further validations using Vero E6 and HuH7 cell lines and hNEC. ( b ) Uninfected cells, infected cells treated with 0.1% DMSO, and infected cells treated with remdesivir were included as controls in the screen. Following treatment with citicoline, pravastatin sodium, tenofovir alafenamide, imatinib mesylate, calcitriol, dexlansoprazole, and prochlorperazine dimaleate, reduced CPE was observed when compared to the DMSO control.

    Techniques Used: Drug discovery, Infection, Control

    Summary of compounds identified from the primary screen that exhibit potential antiviral effects against SARS-CoV-2 infection.
    Figure Legend Snippet: Summary of compounds identified from the primary screen that exhibit potential antiviral effects against SARS-CoV-2 infection.

    Techniques Used: Infection, Reverse Transcription, Reflux

    Validation of primary hits. For validating compounds with activity pre-infection, Vero E6 cells were first pre-treated with increasing concentrations of ( a ) citicoline, ( b ) pravastatin sodium and ( c ) tenofovir alafenamide prior to infection with SARS-CoV-2. Similarly, Huh7 cells were pre-treated with ( d ) citicoline, ( e ) pravastatin sodium and ( f ) tenofovir alafenamide and subsequently infected with SARS-CoV-2. For post-infection treatment validation, Vero E6 cells were infected with SARS-CoV-2 and treated with increasing concentrations of ( g ) imatinib mesylate, ( h ) calcitriol, ( i ) dexlansoprazole and ( j ) prochlorperazine dimaleate. Similarly, HuH7 cells were also infected then treated with a range of concentrations of ( k ) imatinib mesylate, ( l ) calcitriol, ( m ) dexlansoprazole and ( n ) prochlorperazine dimaleate. The primary and secondary exes correspond to the viral titre and relative cell viability, and the dashed line represents the CC 50 cut-off for cell viability. One-way ANOVA and Dunnett’s post-test were used to determine statistical differences, with * denoting p < 0.05, ** denoting p < 0.01 and *** denoting p < 0.001. Error bars represent the standard deviation observed from the means of triplicates performed for both cell viability and dose-dependent inhibition studies.
    Figure Legend Snippet: Validation of primary hits. For validating compounds with activity pre-infection, Vero E6 cells were first pre-treated with increasing concentrations of ( a ) citicoline, ( b ) pravastatin sodium and ( c ) tenofovir alafenamide prior to infection with SARS-CoV-2. Similarly, Huh7 cells were pre-treated with ( d ) citicoline, ( e ) pravastatin sodium and ( f ) tenofovir alafenamide and subsequently infected with SARS-CoV-2. For post-infection treatment validation, Vero E6 cells were infected with SARS-CoV-2 and treated with increasing concentrations of ( g ) imatinib mesylate, ( h ) calcitriol, ( i ) dexlansoprazole and ( j ) prochlorperazine dimaleate. Similarly, HuH7 cells were also infected then treated with a range of concentrations of ( k ) imatinib mesylate, ( l ) calcitriol, ( m ) dexlansoprazole and ( n ) prochlorperazine dimaleate. The primary and secondary exes correspond to the viral titre and relative cell viability, and the dashed line represents the CC 50 cut-off for cell viability. One-way ANOVA and Dunnett’s post-test were used to determine statistical differences, with * denoting p < 0.05, ** denoting p < 0.01 and *** denoting p < 0.001. Error bars represent the standard deviation observed from the means of triplicates performed for both cell viability and dose-dependent inhibition studies.

    Techniques Used: Biomarker Discovery, Activity Assay, Infection, Standard Deviation, Inhibition

    Validation of primary hits in hNECs. Selected primary hits were further validated in hNECs. Cells were either pre-treated with 10 μM citicoline prior to SARS-CoV-2 infection or treated following SARS-CoV-2 infection with 10 μM imatinib mesylate and 10 μM calcitriol. One-way ANOVA and Dunnett’s post-test were used to determine statistical differences, with ** denoting that p < 0.01. Error bars represent the standard deviation observed from the means of triplicates performed for dose-dependent inhibition studies.
    Figure Legend Snippet: Validation of primary hits in hNECs. Selected primary hits were further validated in hNECs. Cells were either pre-treated with 10 μM citicoline prior to SARS-CoV-2 infection or treated following SARS-CoV-2 infection with 10 μM imatinib mesylate and 10 μM calcitriol. One-way ANOVA and Dunnett’s post-test were used to determine statistical differences, with ** denoting that p < 0.01. Error bars represent the standard deviation observed from the means of triplicates performed for dose-dependent inhibition studies.

    Techniques Used: Biomarker Discovery, Infection, Standard Deviation, Inhibition

    SARS-CoV-2 and calcitriol modulate vitamin D Receptor (VDR), 24-hydroxylase (24(OH)ase), and cathelicidin (LL-37) in Vero E6 and Huh7 cells. In the first part of the experiment, Vero E6 and Huh7 cells were either mock-infected with media, or SARS-CoV-2 infected and harvested at indicated timepoints in the absence of calcitriol for baseline VDR, 24(OH)ase, and LL-37 mRNA expression. Vero E6 expression of ( a ) VDR, ( b ) 24(OH)ase and ( c ) LL-37 and Huh7 expression of ( d ) VDR, ( e ) 24(OH)ase and ( f ) LL-37 were as shown. In the second part of the experiment, Vero E6 and Huh7 cells were either mock-infected with media or SARS-CoV-2 for 1 h as above, then treated with 0.01, 0.1, 1, 10 or 20 µM calcitriol following infection. At 48 h post infection, the cells were harvested to check for VDR, 24(OH)ase, and LL-37 mRNA expression. Vero E6 mRNA expression of ( g ) VDR, ( i ) 24(OH)ase and ( k ) LL-37 and Huh7 expression of ( h ) VDR, ( j ) 24(OH)ase and ( l ) LL-37 were as shown. Data are represented as relative quantity to 0.1% DMSO control, or as relative quantity to mock-infected control. One-way ANOVA and Dunnett’s post-test and paired t -test were used to determine statistical differences, with * denoting p < 0.05, ** denoting p < 0.01 and *** denoting p < 0.001. Error bars represent the standard deviation observed from the means of triplicates performed for mRNA expression studies.
    Figure Legend Snippet: SARS-CoV-2 and calcitriol modulate vitamin D Receptor (VDR), 24-hydroxylase (24(OH)ase), and cathelicidin (LL-37) in Vero E6 and Huh7 cells. In the first part of the experiment, Vero E6 and Huh7 cells were either mock-infected with media, or SARS-CoV-2 infected and harvested at indicated timepoints in the absence of calcitriol for baseline VDR, 24(OH)ase, and LL-37 mRNA expression. Vero E6 expression of ( a ) VDR, ( b ) 24(OH)ase and ( c ) LL-37 and Huh7 expression of ( d ) VDR, ( e ) 24(OH)ase and ( f ) LL-37 were as shown. In the second part of the experiment, Vero E6 and Huh7 cells were either mock-infected with media or SARS-CoV-2 for 1 h as above, then treated with 0.01, 0.1, 1, 10 or 20 µM calcitriol following infection. At 48 h post infection, the cells were harvested to check for VDR, 24(OH)ase, and LL-37 mRNA expression. Vero E6 mRNA expression of ( g ) VDR, ( i ) 24(OH)ase and ( k ) LL-37 and Huh7 expression of ( h ) VDR, ( j ) 24(OH)ase and ( l ) LL-37 were as shown. Data are represented as relative quantity to 0.1% DMSO control, or as relative quantity to mock-infected control. One-way ANOVA and Dunnett’s post-test and paired t -test were used to determine statistical differences, with * denoting p < 0.05, ** denoting p < 0.01 and *** denoting p < 0.001. Error bars represent the standard deviation observed from the means of triplicates performed for mRNA expression studies.

    Techniques Used: Infection, Expressing, Control, Standard Deviation

    Western blot analysis of SARS-CoV-2 spike and VDR proteins in Vero E6 cells. ( a ) Vero E6 cells are either mock infected with media (−) or with SARS-CoV-2 (+) for 1 h. After 1 h post-infection, the cells are then either untreated (−) or treated (+) with 20 µM calcitriol for 6-, 24- and 48- hours before harvesting of total cell lysate. ( b ) Densitometry quantitation of protein expression levels is shown as fold changes to beta-actin. Full-length blots are shown in .
    Figure Legend Snippet: Western blot analysis of SARS-CoV-2 spike and VDR proteins in Vero E6 cells. ( a ) Vero E6 cells are either mock infected with media (−) or with SARS-CoV-2 (+) for 1 h. After 1 h post-infection, the cells are then either untreated (−) or treated (+) with 20 µM calcitriol for 6-, 24- and 48- hours before harvesting of total cell lysate. ( b ) Densitometry quantitation of protein expression levels is shown as fold changes to beta-actin. Full-length blots are shown in .

    Techniques Used: Western Blot, Infection, Quantitation Assay, Expressing

    In vivo study of calcitriol treatment. 8-week old K18-hACE2 male and female mice were separated into treatment and non-treatment groups. Treatment study group has been given 3 days pre-treatment and 5 days post-treatment of calcitriol (5 μg/kg) via intraperitoneal injection. The non-treatment group has been given a PBS as a treatment control. Next, 10 3 PFU of SARS-CoV-2 (L, Alpha, and Beta variants) were inoculated through intranasal delivery to all groups after pre-treatment. Physiological parameters: body weight ( a – c ), survival rate ( d – f ), and physiological conditions were monitored after infections. For comparison between individual physiological conditions, 5 dpi infected mice were compared between treatment groups of all three variant infected mice ( g – i ). Scoring of physiological conditions was based on five criteria: appearance of the mouse coat, level of consciousness, activity level, eye condition, and respiratory quality. To compare the severity of damage in mouse tissues after infection, left lung lobes from 4 dpi mice were processed for histological analyses and scored based on six criteria to determine severity ( j – l ). The six criteria are inflammatory cell infiltration, haemorrhage, oedema, bronchial epithelial cell damage, degeneration of alveolar epithelial cells, and parenchymal wall expansion. For viral load determination, right lung, brain, liver, and spleen tissues from the same 4 dpi mice were harvested and homogenised for virus titration ( m – o ). Data is not shown for liver and spleen tissues. Statistical significance is determined with a two-tailed unpaired t -test, with * denoting p < 0.05. Survival group: variant L: n = 6 (treatment and no treatment); Alpha: n = 6 (treatment), n = 4 (no treatment); Beta: n = 6 (treatment), n = 5 (no treatment). 4 dpi group: variant L: n = 7 (treatment and no treatment); Alpha and Beta: n = 5 each (treatment and no treatment). Mock infection, n = 3.
    Figure Legend Snippet: In vivo study of calcitriol treatment. 8-week old K18-hACE2 male and female mice were separated into treatment and non-treatment groups. Treatment study group has been given 3 days pre-treatment and 5 days post-treatment of calcitriol (5 μg/kg) via intraperitoneal injection. The non-treatment group has been given a PBS as a treatment control. Next, 10 3 PFU of SARS-CoV-2 (L, Alpha, and Beta variants) were inoculated through intranasal delivery to all groups after pre-treatment. Physiological parameters: body weight ( a – c ), survival rate ( d – f ), and physiological conditions were monitored after infections. For comparison between individual physiological conditions, 5 dpi infected mice were compared between treatment groups of all three variant infected mice ( g – i ). Scoring of physiological conditions was based on five criteria: appearance of the mouse coat, level of consciousness, activity level, eye condition, and respiratory quality. To compare the severity of damage in mouse tissues after infection, left lung lobes from 4 dpi mice were processed for histological analyses and scored based on six criteria to determine severity ( j – l ). The six criteria are inflammatory cell infiltration, haemorrhage, oedema, bronchial epithelial cell damage, degeneration of alveolar epithelial cells, and parenchymal wall expansion. For viral load determination, right lung, brain, liver, and spleen tissues from the same 4 dpi mice were harvested and homogenised for virus titration ( m – o ). Data is not shown for liver and spleen tissues. Statistical significance is determined with a two-tailed unpaired t -test, with * denoting p < 0.05. Survival group: variant L: n = 6 (treatment and no treatment); Alpha: n = 6 (treatment), n = 4 (no treatment); Beta: n = 6 (treatment), n = 5 (no treatment). 4 dpi group: variant L: n = 7 (treatment and no treatment); Alpha and Beta: n = 5 each (treatment and no treatment). Mock infection, n = 3.

    Techniques Used: In Vivo, Injection, Control, Comparison, Infection, Variant Assay, Activity Assay, Virus, Titration, Two Tailed Test

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    (a) The PRD1 bacteriophage possesses a 14927 bp genome encoding four proteins involved in protein-primed DNA <t>replication:</t> the DNA polymerase P1, the terminal protein P8, and the single-stranded DNA-binding proteins P12 and P19. (b) PRIME comprises four plasmids: pORM ( O rthogonal R eplication M achinery), pOTH ( O rthogonal T ransformation H elper), pORFITR ( O rthogonal R eplicons F lanked by Inverted T erminal R epeats), and lopEG ( L inear O rthogonal P lasmid with E volving G enes). The pORM plasmid carries the genes required for protein-primed DNA replication under control of the inducible P Cin promoter. P12_T2 and P19 are indicated in brackets, as these proteins are dispensable for <t>orthogonal</t> plasmid replication but are present in the pORM_P12_T2 construct. pORM additionally contains the broad-host-range pBBR1 origin of replication, enabling propagation in a wide range of bacterial hosts. The pOTH plasmid encodes accessory proteins facilitating lopEG transformation. Owing to their toxicity, these proteins are expressed under control of the inducible P BAD promoter. The temperature-sensitive pSC101(ts) origin enables efficient curing of pOTH following lopEG establishment. The pORFITR plasmid facilitates cloning and production of lopEG plasmids. It contains two inverted terminal repeat (ITR) regions flanked by MlyI recognition sites. As a Type IIS restriction enzyme, MlyI cleaves outside of its recognition sequence, enabling blunt cleavage precisely at the edges of the ITRs. The R6K conditional origin of replication prevents plasmid propagation in cells lacking the Pir protein, allowing direct transformation of lopEG plasmids without gel purification. (c) Workflow for establishment of orthogonal plasmids in E. coli . (1) Cells are first transformed with pORM and pOTH. (2) Expression of the replication proteins is induced, and the temperature is increased prior to lopEG transformation to inhibit further replication of pOTH. (3) lopEG plasmids are electroporated into competent cells expressing the PRD1 replication machinery. Orthogonal plasmid copy number can subsequently be tuned using the OHC14-HSL inducer. (d) PRIME enables continuous directed evolution experiments. Replacement of the wild-type P1 polymerase (green) with an error-prone variant (red) promotes the generation of DNA libraries directly in vivo without the need for repeated manual intervention. Iterative cycles of diversification and selection subsequently enable the rapid evolution of constructs with improved properties.
    Oitv Replicates, supplied by Sawai Pharmaceutical, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    conventional non replicating mrna based therapeutics  (Moderna)
    86
    Moderna conventional non replicating mrna based therapeutics
    (a) The PRD1 bacteriophage possesses a 14927 bp genome encoding four proteins involved in protein-primed DNA <t>replication:</t> the DNA polymerase P1, the terminal protein P8, and the single-stranded DNA-binding proteins P12 and P19. (b) PRIME comprises four plasmids: pORM ( O rthogonal R eplication M achinery), pOTH ( O rthogonal T ransformation H elper), pORFITR ( O rthogonal R eplicons F lanked by Inverted T erminal R epeats), and lopEG ( L inear O rthogonal P lasmid with E volving G enes). The pORM plasmid carries the genes required for protein-primed DNA replication under control of the inducible P Cin promoter. P12_T2 and P19 are indicated in brackets, as these proteins are dispensable for <t>orthogonal</t> plasmid replication but are present in the pORM_P12_T2 construct. pORM additionally contains the broad-host-range pBBR1 origin of replication, enabling propagation in a wide range of bacterial hosts. The pOTH plasmid encodes accessory proteins facilitating lopEG transformation. Owing to their toxicity, these proteins are expressed under control of the inducible P BAD promoter. The temperature-sensitive pSC101(ts) origin enables efficient curing of pOTH following lopEG establishment. The pORFITR plasmid facilitates cloning and production of lopEG plasmids. It contains two inverted terminal repeat (ITR) regions flanked by MlyI recognition sites. As a Type IIS restriction enzyme, MlyI cleaves outside of its recognition sequence, enabling blunt cleavage precisely at the edges of the ITRs. The R6K conditional origin of replication prevents plasmid propagation in cells lacking the Pir protein, allowing direct transformation of lopEG plasmids without gel purification. (c) Workflow for establishment of orthogonal plasmids in E. coli . (1) Cells are first transformed with pORM and pOTH. (2) Expression of the replication proteins is induced, and the temperature is increased prior to lopEG transformation to inhibit further replication of pOTH. (3) lopEG plasmids are electroporated into competent cells expressing the PRD1 replication machinery. Orthogonal plasmid copy number can subsequently be tuned using the OHC14-HSL inducer. (d) PRIME enables continuous directed evolution experiments. Replacement of the wild-type P1 polymerase (green) with an error-prone variant (red) promotes the generation of DNA libraries directly in vivo without the need for repeated manual intervention. Iterative cycles of diversification and selection subsequently enable the rapid evolution of constructs with improved properties.
    Conventional Non Replicating Mrna Based Therapeutics, supplied by Moderna, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    replication evidence  (Hasegawa Co Ltd)
    86
    Hasegawa Co Ltd replication evidence
    (a) The PRD1 bacteriophage possesses a 14927 bp genome encoding four proteins involved in protein-primed DNA <t>replication:</t> the DNA polymerase P1, the terminal protein P8, and the single-stranded DNA-binding proteins P12 and P19. (b) PRIME comprises four plasmids: pORM ( O rthogonal R eplication M achinery), pOTH ( O rthogonal T ransformation H elper), pORFITR ( O rthogonal R eplicons F lanked by Inverted T erminal R epeats), and lopEG ( L inear O rthogonal P lasmid with E volving G enes). The pORM plasmid carries the genes required for protein-primed DNA replication under control of the inducible P Cin promoter. P12_T2 and P19 are indicated in brackets, as these proteins are dispensable for <t>orthogonal</t> plasmid replication but are present in the pORM_P12_T2 construct. pORM additionally contains the broad-host-range pBBR1 origin of replication, enabling propagation in a wide range of bacterial hosts. The pOTH plasmid encodes accessory proteins facilitating lopEG transformation. Owing to their toxicity, these proteins are expressed under control of the inducible P BAD promoter. The temperature-sensitive pSC101(ts) origin enables efficient curing of pOTH following lopEG establishment. The pORFITR plasmid facilitates cloning and production of lopEG plasmids. It contains two inverted terminal repeat (ITR) regions flanked by MlyI recognition sites. As a Type IIS restriction enzyme, MlyI cleaves outside of its recognition sequence, enabling blunt cleavage precisely at the edges of the ITRs. The R6K conditional origin of replication prevents plasmid propagation in cells lacking the Pir protein, allowing direct transformation of lopEG plasmids without gel purification. (c) Workflow for establishment of orthogonal plasmids in E. coli . (1) Cells are first transformed with pORM and pOTH. (2) Expression of the replication proteins is induced, and the temperature is increased prior to lopEG transformation to inhibit further replication of pOTH. (3) lopEG plasmids are electroporated into competent cells expressing the PRD1 replication machinery. Orthogonal plasmid copy number can subsequently be tuned using the OHC14-HSL inducer. (d) PRIME enables continuous directed evolution experiments. Replacement of the wild-type P1 polymerase (green) with an error-prone variant (red) promotes the generation of DNA libraries directly in vivo without the need for repeated manual intervention. Iterative cycles of diversification and selection subsequently enable the rapid evolution of constructs with improved properties.
    Replication Evidence, supplied by Hasegawa Co Ltd, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    replication codes  (Mendeley Ltd)
    86
    Mendeley Ltd replication codes
    (a) The PRD1 bacteriophage possesses a 14927 bp genome encoding four proteins involved in protein-primed DNA <t>replication:</t> the DNA polymerase P1, the terminal protein P8, and the single-stranded DNA-binding proteins P12 and P19. (b) PRIME comprises four plasmids: pORM ( O rthogonal R eplication M achinery), pOTH ( O rthogonal T ransformation H elper), pORFITR ( O rthogonal R eplicons F lanked by Inverted T erminal R epeats), and lopEG ( L inear O rthogonal P lasmid with E volving G enes). The pORM plasmid carries the genes required for protein-primed DNA replication under control of the inducible P Cin promoter. P12_T2 and P19 are indicated in brackets, as these proteins are dispensable for <t>orthogonal</t> plasmid replication but are present in the pORM_P12_T2 construct. pORM additionally contains the broad-host-range pBBR1 origin of replication, enabling propagation in a wide range of bacterial hosts. The pOTH plasmid encodes accessory proteins facilitating lopEG transformation. Owing to their toxicity, these proteins are expressed under control of the inducible P BAD promoter. The temperature-sensitive pSC101(ts) origin enables efficient curing of pOTH following lopEG establishment. The pORFITR plasmid facilitates cloning and production of lopEG plasmids. It contains two inverted terminal repeat (ITR) regions flanked by MlyI recognition sites. As a Type IIS restriction enzyme, MlyI cleaves outside of its recognition sequence, enabling blunt cleavage precisely at the edges of the ITRs. The R6K conditional origin of replication prevents plasmid propagation in cells lacking the Pir protein, allowing direct transformation of lopEG plasmids without gel purification. (c) Workflow for establishment of orthogonal plasmids in E. coli . (1) Cells are first transformed with pORM and pOTH. (2) Expression of the replication proteins is induced, and the temperature is increased prior to lopEG transformation to inhibit further replication of pOTH. (3) lopEG plasmids are electroporated into competent cells expressing the PRD1 replication machinery. Orthogonal plasmid copy number can subsequently be tuned using the OHC14-HSL inducer. (d) PRIME enables continuous directed evolution experiments. Replacement of the wild-type P1 polymerase (green) with an error-prone variant (red) promotes the generation of DNA libraries directly in vivo without the need for repeated manual intervention. Iterative cycles of diversification and selection subsequently enable the rapid evolution of constructs with improved properties.
    Replication Codes, supplied by Mendeley Ltd, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96 pin replicator  (Boekel Scientific)
    86
    Boekel Scientific 96 pin replicator
    (a) The PRD1 bacteriophage possesses a 14927 bp genome encoding four proteins involved in protein-primed DNA <t>replication:</t> the DNA polymerase P1, the terminal protein P8, and the single-stranded DNA-binding proteins P12 and P19. (b) PRIME comprises four plasmids: pORM ( O rthogonal R eplication M achinery), pOTH ( O rthogonal T ransformation H elper), pORFITR ( O rthogonal R eplicons F lanked by Inverted T erminal R epeats), and lopEG ( L inear O rthogonal P lasmid with E volving G enes). The pORM plasmid carries the genes required for protein-primed DNA replication under control of the inducible P Cin promoter. P12_T2 and P19 are indicated in brackets, as these proteins are dispensable for <t>orthogonal</t> plasmid replication but are present in the pORM_P12_T2 construct. pORM additionally contains the broad-host-range pBBR1 origin of replication, enabling propagation in a wide range of bacterial hosts. The pOTH plasmid encodes accessory proteins facilitating lopEG transformation. Owing to their toxicity, these proteins are expressed under control of the inducible P BAD promoter. The temperature-sensitive pSC101(ts) origin enables efficient curing of pOTH following lopEG establishment. The pORFITR plasmid facilitates cloning and production of lopEG plasmids. It contains two inverted terminal repeat (ITR) regions flanked by MlyI recognition sites. As a Type IIS restriction enzyme, MlyI cleaves outside of its recognition sequence, enabling blunt cleavage precisely at the edges of the ITRs. The R6K conditional origin of replication prevents plasmid propagation in cells lacking the Pir protein, allowing direct transformation of lopEG plasmids without gel purification. (c) Workflow for establishment of orthogonal plasmids in E. coli . (1) Cells are first transformed with pORM and pOTH. (2) Expression of the replication proteins is induced, and the temperature is increased prior to lopEG transformation to inhibit further replication of pOTH. (3) lopEG plasmids are electroporated into competent cells expressing the PRD1 replication machinery. Orthogonal plasmid copy number can subsequently be tuned using the OHC14-HSL inducer. (d) PRIME enables continuous directed evolution experiments. Replacement of the wild-type P1 polymerase (green) with an error-prone variant (red) promotes the generation of DNA libraries directly in vivo without the need for repeated manual intervention. Iterative cycles of diversification and selection subsequently enable the rapid evolution of constructs with improved properties.
    96 Pin Replicator, supplied by Boekel Scientific, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    replication  (Bavarian Nordic)
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    Bavarian Nordic replication
    (a) The PRD1 bacteriophage possesses a 14927 bp genome encoding four proteins involved in protein-primed DNA <t>replication:</t> the DNA polymerase P1, the terminal protein P8, and the single-stranded DNA-binding proteins P12 and P19. (b) PRIME comprises four plasmids: pORM ( O rthogonal R eplication M achinery), pOTH ( O rthogonal T ransformation H elper), pORFITR ( O rthogonal R eplicons F lanked by Inverted T erminal R epeats), and lopEG ( L inear O rthogonal P lasmid with E volving G enes). The pORM plasmid carries the genes required for protein-primed DNA replication under control of the inducible P Cin promoter. P12_T2 and P19 are indicated in brackets, as these proteins are dispensable for <t>orthogonal</t> plasmid replication but are present in the pORM_P12_T2 construct. pORM additionally contains the broad-host-range pBBR1 origin of replication, enabling propagation in a wide range of bacterial hosts. The pOTH plasmid encodes accessory proteins facilitating lopEG transformation. Owing to their toxicity, these proteins are expressed under control of the inducible P BAD promoter. The temperature-sensitive pSC101(ts) origin enables efficient curing of pOTH following lopEG establishment. The pORFITR plasmid facilitates cloning and production of lopEG plasmids. It contains two inverted terminal repeat (ITR) regions flanked by MlyI recognition sites. As a Type IIS restriction enzyme, MlyI cleaves outside of its recognition sequence, enabling blunt cleavage precisely at the edges of the ITRs. The R6K conditional origin of replication prevents plasmid propagation in cells lacking the Pir protein, allowing direct transformation of lopEG plasmids without gel purification. (c) Workflow for establishment of orthogonal plasmids in E. coli . (1) Cells are first transformed with pORM and pOTH. (2) Expression of the replication proteins is induced, and the temperature is increased prior to lopEG transformation to inhibit further replication of pOTH. (3) lopEG plasmids are electroporated into competent cells expressing the PRD1 replication machinery. Orthogonal plasmid copy number can subsequently be tuned using the OHC14-HSL inducer. (d) PRIME enables continuous directed evolution experiments. Replacement of the wild-type P1 polymerase (green) with an error-prone variant (red) promotes the generation of DNA libraries directly in vivo without the need for repeated manual intervention. Iterative cycles of diversification and selection subsequently enable the rapid evolution of constructs with improved properties.
    Replication, supplied by Bavarian Nordic, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    replication  (MedChemExpress)
    94
    MedChemExpress replication
    (a) The PRD1 bacteriophage possesses a 14927 bp genome encoding four proteins involved in protein-primed DNA <t>replication:</t> the DNA polymerase P1, the terminal protein P8, and the single-stranded DNA-binding proteins P12 and P19. (b) PRIME comprises four plasmids: pORM ( O rthogonal R eplication M achinery), pOTH ( O rthogonal T ransformation H elper), pORFITR ( O rthogonal R eplicons F lanked by Inverted T erminal R epeats), and lopEG ( L inear O rthogonal P lasmid with E volving G enes). The pORM plasmid carries the genes required for protein-primed DNA replication under control of the inducible P Cin promoter. P12_T2 and P19 are indicated in brackets, as these proteins are dispensable for <t>orthogonal</t> plasmid replication but are present in the pORM_P12_T2 construct. pORM additionally contains the broad-host-range pBBR1 origin of replication, enabling propagation in a wide range of bacterial hosts. The pOTH plasmid encodes accessory proteins facilitating lopEG transformation. Owing to their toxicity, these proteins are expressed under control of the inducible P BAD promoter. The temperature-sensitive pSC101(ts) origin enables efficient curing of pOTH following lopEG establishment. The pORFITR plasmid facilitates cloning and production of lopEG plasmids. It contains two inverted terminal repeat (ITR) regions flanked by MlyI recognition sites. As a Type IIS restriction enzyme, MlyI cleaves outside of its recognition sequence, enabling blunt cleavage precisely at the edges of the ITRs. The R6K conditional origin of replication prevents plasmid propagation in cells lacking the Pir protein, allowing direct transformation of lopEG plasmids without gel purification. (c) Workflow for establishment of orthogonal plasmids in E. coli . (1) Cells are first transformed with pORM and pOTH. (2) Expression of the replication proteins is induced, and the temperature is increased prior to lopEG transformation to inhibit further replication of pOTH. (3) lopEG plasmids are electroporated into competent cells expressing the PRD1 replication machinery. Orthogonal plasmid copy number can subsequently be tuned using the OHC14-HSL inducer. (d) PRIME enables continuous directed evolution experiments. Replacement of the wild-type P1 polymerase (green) with an error-prone variant (red) promotes the generation of DNA libraries directly in vivo without the need for repeated manual intervention. Iterative cycles of diversification and selection subsequently enable the rapid evolution of constructs with improved properties.
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    Image Search Results


    (a) The PRD1 bacteriophage possesses a 14927 bp genome encoding four proteins involved in protein-primed DNA replication: the DNA polymerase P1, the terminal protein P8, and the single-stranded DNA-binding proteins P12 and P19. (b) PRIME comprises four plasmids: pORM ( O rthogonal R eplication M achinery), pOTH ( O rthogonal T ransformation H elper), pORFITR ( O rthogonal R eplicons F lanked by Inverted T erminal R epeats), and lopEG ( L inear O rthogonal P lasmid with E volving G enes). The pORM plasmid carries the genes required for protein-primed DNA replication under control of the inducible P Cin promoter. P12_T2 and P19 are indicated in brackets, as these proteins are dispensable for orthogonal plasmid replication but are present in the pORM_P12_T2 construct. pORM additionally contains the broad-host-range pBBR1 origin of replication, enabling propagation in a wide range of bacterial hosts. The pOTH plasmid encodes accessory proteins facilitating lopEG transformation. Owing to their toxicity, these proteins are expressed under control of the inducible P BAD promoter. The temperature-sensitive pSC101(ts) origin enables efficient curing of pOTH following lopEG establishment. The pORFITR plasmid facilitates cloning and production of lopEG plasmids. It contains two inverted terminal repeat (ITR) regions flanked by MlyI recognition sites. As a Type IIS restriction enzyme, MlyI cleaves outside of its recognition sequence, enabling blunt cleavage precisely at the edges of the ITRs. The R6K conditional origin of replication prevents plasmid propagation in cells lacking the Pir protein, allowing direct transformation of lopEG plasmids without gel purification. (c) Workflow for establishment of orthogonal plasmids in E. coli . (1) Cells are first transformed with pORM and pOTH. (2) Expression of the replication proteins is induced, and the temperature is increased prior to lopEG transformation to inhibit further replication of pOTH. (3) lopEG plasmids are electroporated into competent cells expressing the PRD1 replication machinery. Orthogonal plasmid copy number can subsequently be tuned using the OHC14-HSL inducer. (d) PRIME enables continuous directed evolution experiments. Replacement of the wild-type P1 polymerase (green) with an error-prone variant (red) promotes the generation of DNA libraries directly in vivo without the need for repeated manual intervention. Iterative cycles of diversification and selection subsequently enable the rapid evolution of constructs with improved properties.

    Journal: bioRxiv

    Article Title: PRIME: A modular plasmid-based platform for continuous in vivo evolution across bacteria

    doi: 10.64898/2026.09.28.754915

    Figure Lengend Snippet: (a) The PRD1 bacteriophage possesses a 14927 bp genome encoding four proteins involved in protein-primed DNA replication: the DNA polymerase P1, the terminal protein P8, and the single-stranded DNA-binding proteins P12 and P19. (b) PRIME comprises four plasmids: pORM ( O rthogonal R eplication M achinery), pOTH ( O rthogonal T ransformation H elper), pORFITR ( O rthogonal R eplicons F lanked by Inverted T erminal R epeats), and lopEG ( L inear O rthogonal P lasmid with E volving G enes). The pORM plasmid carries the genes required for protein-primed DNA replication under control of the inducible P Cin promoter. P12_T2 and P19 are indicated in brackets, as these proteins are dispensable for orthogonal plasmid replication but are present in the pORM_P12_T2 construct. pORM additionally contains the broad-host-range pBBR1 origin of replication, enabling propagation in a wide range of bacterial hosts. The pOTH plasmid encodes accessory proteins facilitating lopEG transformation. Owing to their toxicity, these proteins are expressed under control of the inducible P BAD promoter. The temperature-sensitive pSC101(ts) origin enables efficient curing of pOTH following lopEG establishment. The pORFITR plasmid facilitates cloning and production of lopEG plasmids. It contains two inverted terminal repeat (ITR) regions flanked by MlyI recognition sites. As a Type IIS restriction enzyme, MlyI cleaves outside of its recognition sequence, enabling blunt cleavage precisely at the edges of the ITRs. The R6K conditional origin of replication prevents plasmid propagation in cells lacking the Pir protein, allowing direct transformation of lopEG plasmids without gel purification. (c) Workflow for establishment of orthogonal plasmids in E. coli . (1) Cells are first transformed with pORM and pOTH. (2) Expression of the replication proteins is induced, and the temperature is increased prior to lopEG transformation to inhibit further replication of pOTH. (3) lopEG plasmids are electroporated into competent cells expressing the PRD1 replication machinery. Orthogonal plasmid copy number can subsequently be tuned using the OHC14-HSL inducer. (d) PRIME enables continuous directed evolution experiments. Replacement of the wild-type P1 polymerase (green) with an error-prone variant (red) promotes the generation of DNA libraries directly in vivo without the need for repeated manual intervention. Iterative cycles of diversification and selection subsequently enable the rapid evolution of constructs with improved properties.

    Article Snippet: The YFP gene was then replaced with the operon encoding for the orthogonal replication machinery by digestion with BsaI-HF-v2 and KpnI-HF restriction enzymes (New England Biolabs) and ligation with a similarly digested insert, yielding pORM_P12_P19.

    Techniques: DNA Binding Assay, Plasmid Preparation, Control, Construct, Transformation Assay, Toxicity, Cloning, Sequencing, Gel Purification, Expressing, Variant Assay, In Vivo, Selection, Protein Binding