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Research Solutions for Coronavirus

Coronaviruses are named for the crown-like spikes on their surface. There are four main sub-groupings of coronaviruses, known as alpha, beta, gamma, and delta. Novel coronavirus (SARS-CoV-2) is a beta coronavirus. SARS-CoV-2 is a positive-sense, single-stranded RNA coronavirus that causes COVID-19, the infectious disease that can cause an acute respiratory infection. Studies show that the SARS-CoV-2 S protein retains sufficient affinity to the cellular Angiotensin converting enzyme 2 (ACE2) protein, and likely uses ACE2 protein as a receptor for cellular entry.

There is currently no specific medicine or treatment for diseases caused by SARS-CoV-2. To identify antiviral candidates, scientists are focused on drug repurposing and antiviral molecules screening from chemical libraries. We offer efficient compound libraries to assist in the global efforts to develop novel drug candidates, such as FDA-approved Drug Library and Anti-Virus Compound Library. We also offer reverse transcription and qPCR series to help efficiently detect SARS-CoV-2, and mRNA in vitro synthesis platform to support the development of mRNA vaccines.

1. Screening Libraries

Cat.No. Product Name Information
L1021 FDA-approved Drug Library 1971 FDA-approved drugs
L1022 Bioactive Compound Library 3317 bioactive compounds
L1022P Bioactive Compound Library Plus 4417 bioactive compounds
L1050 Anti-Virus Compound Library 264 anti-virus compounds
L1039 Natural Product Library 550 natural products
L1039P Natural Product Library Plus 1170 natural products
L1027 Anti-infection Compound Library 367 anti-infection small molecules
L1027P Anti-infection Compound Library Plus 663 anti-infection small molecules
L1035 Protease Inhibitor Library 825 protease inhibitors
L1026 Neuronal Signaling Library 556 neuronal signaling-related small molecules
L1026P Neuronal Signaling Compound Library Plus 948 neuronal signaling-related small molecules
L1024 Kinase Inhibitor Library 796 kinase inhibitors
L1023 Anti-cancer Compound Library 1164 anti-cancer compounds
L1023P Anti-cancer Compound Library Plus 1777 anti-cancer compounds
L1025 GPCR Compound Library 677 GPCR-related small molecules
L1029 Epigenetics Compound Library 328 epigenetics-related small molecules
L1034 PI3K/Akt/mTOR Compound Library 178 PI3K/Akt/mTOR inhibitors
L1041 JAK/STAT Compound Library 109 JAK/STAT inhibitors
L1028 Tyrosine Kinase Inhibitor Library 369 tyrosine kinase inhibitors
L1031 Autophagy Compound Library 486 autophagy-related compounds
L1031P Autophagy Compound Library Plus 837 autophagy-related compounds
L1032 Metabolism-related Compound Library 493 metabolism-related compounds
L1032P Metabolism-related Compound Library Plus 1205 metabolism-related compounds
L1036 Apoptosis Compound Library 643 apoptosis-related compounds
L1038 Histone Modification Library 157 histone modification-related compounds
L1040 Stem Cell Compound Library 169 stem cell-related compounds
L1040P Stem Cell Compound Library Plus 280 stem cell-related compounds
L1042 Immunology/Inflammation Compound Library 295 immunology/inflammation-related small molecules
L1042P Immunology/Inflammation Compound Library Plus 806 immunology/inflammation-related small molecules
L1043 MAPK Inhibitor Library 117 MAPK inhibitors
L1048 Inhibitor Library 2525 inhibitors
L1033 DNA Damage/DNA Repair Library 481 DNA Damage/DNA repair-related compounds
L1037 Cell Cycle Library 449 cell cycle-related compounds
L1045 TGF-beta/Smad Compound Library 88 TGF-beta/Smad inhibitors
L1030 Ion Channel Compound Library 199 ion channel-related compounds
L1030P Ion Channel Compound Library Plus 350 ion channel-related compounds
L1044 NF-κB Signaling Library 73 NF-κB inhibitors
L1044P NF-κB Signaling Compound Library Plus 178 NF-κB inhibitors
L1046 Anti-diabetic Compound Library 110 anti-diabetic small molecules
L1047 Angiogenesis Library 84 angiogenesis-related small molecules
L1049 Ubiquitination Compound Library 144 ubiquitination compounds

We also offer customized screening libraries that allow you to choose the compounds, the sizes, the quantities, the format (powder or solution) and plate map you want in the library.

2. Reverse Transcriptase, qPCR Series

qPCR amplification curve of SARS-CoV-2 S gene. The initial number of copies of template DNA: 10/20/200 copies. The DNA was quantified using 2X SYBR Green qPCR Master Mix (Cat. No. K1070).

qPCR amplification curve of human β-Action. RNA was extracted from 293A cells and diluted to 7 different concentrations (1μg/μl, 100ng/μl, 10ng/ml, 1ng/μl, 100pg/μl, 10pg/μl, 1pg/μl). Reverse transcription was performed using First-Strand cDNA Synthesis SuperMix (Cat. No. K1073) to obtain cDNA products. The cDNA was then quantified using 2X SYBR Green qPCR Master Mix (Cat. No. K1070).

Cat.No. Product Name Information
K1070 2×SYBR Green qPCR Master Mix 2X PreMix for quantifying target DNA or cDNA
K1071 Reverse Transcriptase Thermally stable reverse transcriptase used to synthesize complementary DNA (cDNA) from an RNA template
K1072 First-Strand cDNA Synthesis Kit Synthesize first-strand cDNA from purified poly(A)+ or total RNA
K1073 First-Strand cDNA Synthesis SuperMix Reverse transcription reaction premixed solution for efficient synthesis of first-strand cDNA
K1074 RT SuperMix for qPCR High efficiency reverse transcription reaction premixed solution for two-step RT-qPCR method
K1046 RNase Inhibitor, Murine RNase Inhibitor, Murine specifically inhibits RNases A, B and C to protect RNA from degradation

3. RNA In Vitro Synthesis Platform

An overview of the workflow developing mRNA vaccines against 2019-nCoV. Both receptor-binding domain of the spike protein (S-RBD) and Virus like particles (VLPs) were chosen as the antigen. Strategy one: Based on the mRNA in vitro transcription platform, SARS-CoV-2 S, M, E mRNA were synthesized in vitro and individually transfected into 293T cells by using lipofectamine 2000. Virus like particles (VLPs) were produced and used as the presenting antigen. VLPs synthesized by the host cells have the same posttranslational modifications as the native virus, which is an important factor determining the validity of an antigen. Strategy two: For the S-RBD antigen, the RBD domain of the S protein was also expressed in cells using in vitro transcription (IVT) mRNA. (Ref.1)

VLPs were visualized under an electron microscope. We observed particles with striking features of coronavirus. The outline of the envelope for most particles was clear, the spikes were visible. The average size of the particle is 70nm in diameter for the membrane envelope and 90nm when including the spikes, consistent with reported native 2019-nCoV virus. (Ref.1)

The trimeric structure of the extra-vesicular domain of the spike protein from SARS-CoV. The receptor-binding domain (RBD) was used as the antigen. Use mRNA to express the receptor-binding domain of the spike protein (S-RBD). The S glycoprotein is responsible for binding to the receptor through its RBD, enabling the virus to enter into target cells by fusing with cell membranes. The western blot analysis was used to confirm the expression of target proteins. (Ref.1)

Reference:

1.Towards an effective mRNA vaccine against 2019-nCoV: demonstration of virus-like particles expressed from an modified mRNA cocktail.

Custom mRNA Synthesis

Affordable Cost, High Yields

APExBIO offers affordable custom synthesis of mRNA and long RNA (up to multiple kilobases) with a wide array of modification services at scales ranging from micrograms to milligrams. The mRNA can be generated from DNA templates provided by our customers or we can provide a full service from the ground up. We provide mCAP, ARCA and EZ Cap (equal to CleanCap) capping or modified nucleotides implication for all our standard mRNA transcripts.

For ordering and more information, please download this form (Custom mRNA Synthesis Questionnaire.PDF) and send it via email ([email protected]). Our support team will review and provide a quotation soon.

Modified Nucleotide-containing mRNA Synthesis

In Vitro Synthesis of mRNA (In vitro transcription, IVT)

A 7-methyl guanosine (m7G) cap structure at the 5′ end and a poly(A) tail at the 3′ end are required for mRNA to be translated efficiently in vitro. Capped mRNAs are synthesized by co-transcriptional incorporation of Anti-Reverse Cap Analog (ARCA) via T7 RNA Polymerase. DNase I is used to remove the template DNA, so Poly(A) Polymerase can attach poly(A) tail to capped mRNA. 5-Methyl-CTP, Pseudo-UTP and other modified nucleotides can also be incorporated into mRNA. Synthetic mRNAs are applicable in cell transfection, microinjection, in vitro translation and RNA vaccines etc.

Our custom synthesis mRNA covers a wide range of applications:

  • mRNA for genome editing, e.g. Zinc-finger Nuclease mRNA, TALEN mRNA, Cas9 mRNA and Recombinase mRNA.
  • Reporter gene mRNA, such as EGFP mRNA and Luc mRNA, for fluorescence microscopy, flow cytometry and bioluminescent imaging.
  • Reprogramming mRNA, i.e mRNA for non-integrating generation of iPSC.

Validation:

Related Products
Cat.No. Product Name Cat.No. Product Name
B8175 ARCA B8174 mCAP
B7972 Pseudo-UTP B7967 5-Methyl-CTP
B8061 5-Methoxy-UTP K1046 RNase Inhibitor, Murine
K1047 HyperScribe™ T7 High Yield RNA Synthesis Kit K1053 HyperScribe™ Poly (A) Tailing Kit
K1060 HyperScribe™ T7 High Yield Fluorescein RNA Labeling Kit K1061 HyperScribe™ T7 High Yield Cy3 RNA Labeling Kit
K1062 HyperScribe™ T7 High Yield Cy5 RNA Labeling Kit K1063 HyperScribe™ All in One mRNA Synthesis Kit (ARCA, T7, poly(A))
K1064 HyperScribe™ All in One mRNA Synthesis Kit Plus 1 (ARCA, 5mCTP, ψUTP, T7, poly(A)) K1065 HyperScribe™ All in One mRNA Synthesis Kit Plus 2 (ARCA, 5-moUTP, T7, poly(A))
K1066 HyperScribe™ All in One mRNA Synthesis Kit II (EZ Cap Reagent AG (3' OMe), T7, poly(A)) K1067 HyperScribe™ All in One mRNA Synthesis Kit II Plus 1 (EZ Cap Reagent AG (3' OMe), 5mCTP, ψUTP, T7, poly(A))
K1068 HyperScribe™ All in One mRNA Synthesis Kit II Plus 2 (EZ Cap Reagent AG (3' OMe), 5-moUTP, T7, poly(A))

mRNA Purification

mRNAs transcribed in vitro by T7 RNA polymerase may contain various contaminants, such as short RNAs produced by abortive initiation events, double-stranded (ds)RNAs generated by self-complementary 3’extension, as well as unincorporated nucleoside triphosphates, small abortive transcripts and plasmid template. Certain RNA sequences even induce high levels immunogenicity.

APExBIO offers purification service to remove the contaminants of modified nucleotide-containing mRNA, thus increase the processing efficiency for downstream applications.

Silica-gel Membrane Spin Column Purification:

It is a solid phase extraction technique for fast nucleic acid purification. mRNA can be bound to solid phase of silica-gel membranes under certain conditions, with subsequent washing and elution steps in water or TE pH 7. This method eliminates most proteins, DNA and NTPs.

HPLC purification by ÄKTA avant system:

mRNA can be purified by HPLC (ÄKTA avant system) using column matrix of alkylated non-porous polystyrene-divinylbenzene copolymer microspheres and optimized buffer system, followed by mRNA analyses and mRNA isolation from column fractions.

HPLC purification removes dsRNA and other contaminants from in vitro synthesized modified nucleotide-containing mRNAs, yielding mRNA with the high level of translation without generation of immunogenicity or RNA sensor activation.

mRNA and long RNA products

APExBIO supplies the best quality mRNA and long RNA. This new product lines involve custom synthesis of mRNA and long RNA (up to multiple kilobases) with a wide array of modification services at scales ranging from micrograms to milligrams. The mRNA can be generated from DNA templates provided by our customers or we can provide a full service from the ground up. We offer mCAP or ARCA capping or modified nucleotides implication for all our standard mRNA transcripts.

All of our mRNA products offer:

  • Incorporates an anti-reverse cap analog (ARCA) into the transcript to increase translation efficiency
  • Reduces host cell immune response and enhances stability by incorporating modified nucleotides (5mCTP and ψUTP) and a poly(A) tail
  • Degrades the DNA template after RNA synthesis with DNase
  • Removes the 5’ triphosphates at the end of the RNA with phosphatase to further reduce innate immune responses in mammalian cells
  • Employs a robust clean-up spin column system that delivers high yields of mRNAs that are ready for most downstream applications

Validation:

Related Products
Cat.No. Product Name Cat.No. Product Name
R1001 ARCA EGFP mRNA R1002 ARCA EGFP mRNA (5mCTP, ψUTP)
R1003 mCAP EGFP mRNA R1004 mCAP EGFP mRNA (5mCTP, ψUTP)
R1005 Firefly Luciferase mRNA (ARCA, 5mCTP, ψUTP) R1006 SpCas9 mRNA (ARCA, 5mCTP, ψUTP)
R1007 ARCA EGFP mRNA (5-moUTP) R1008 ARCA Cy3 EGFP mRNA (5-moUTP)
R1009 ARCA Cy5 EGFP mRNA (5-moUTP) R1010 EZ Cap™ Cy5 Firefly Luciferase mRNA (5-moUTP)
R1011 EZ Cap™ Cy5 EGFP mRNA (5-moUTP) R1012 Firefly Luciferase mRNA (ARCA, 5-moUTP)
R1013 EZ Cap™ Firefly Luciferase mRNA (5-moUTP) R1014 ARCA Cas9 5-moUTP
R1015 EZ Cap™ Cas9 5-moUTP R1016 EZ Cap™ EGFP mRNA (5-moUTP)
R1017 EZ Cap™ mCherry mRNA (5mCTP, ψUTP) R1018 EZ Cap™ Firefly Luciferase mRNA
B8178 EZ Cap™ Reagent AG (3' OMe)

Antibody Drugs and Vaccine Development Related Products

Cat.No. Product Name Information
B8293 ddhCTP Chain terminator for the RNA-dependent RNA polymerases. Novel antiviral nucleotide molecules.
B8362 2,3-cGAMP Immune adjuvant, can specifically activate STING pathway, stimulate innate immunity and accelerate antibody production.
BC1001 Human B Cell Culture and Expansion Kit Used for activation and expansion of human B cells.
BC1002 3T3-msCD40L Cell Lines 3T3-CD40L cells can be co-cultured with B cells to aid B cell expansion.