ApexBio COVID-19

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. We provide reverse transcription and qPCR series to help efficiently detect SARS-CoV-2, and also provide mRNA in vitro synthesis platform for mRNA vaccines development.

1. 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. Viral RNA was extracted from samples derived from patients with SARS-CoV-2. Reverse transcription was performed using reverse transcriptase (Cat. No. K1071) to obtain cDNA products. The cDNA was then quantified using 2X SYBR Green qPCR Master Mix (Cat. No. K1072)

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
K1046	RNase Inhibitor, Murine	RNase Inhibitor, Murine specifically inhibits RNases A, B and C to protect RNA from degradation

2. 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 contact sales@stratech.co.uk. Our support team will review and provide a quotation soon.

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:

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 ?KTA avant system:

mRNA can be purified by HPLC (?KTA 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:

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.

ApexBio COVID-19

Research Solutions for Coronavirus

Custom mRNA Synthesis

Tech Contact

General Contact

SUPPORT

PRODUCT

DELIVERY

ApexBio COVID-19

Research Solutions for Coronavirus

Custom mRNA Synthesis

Your first choice for scientific solutions

Tech Contact

General Contact

SUPPORT

PRODUCT

DELIVERY