Did you know that mRNA vaccines represent the latest breakthrough in nucleic acid vaccine development? They build upon the success of inactivated and attenuated live vaccines (first generation) as well as subunit vaccines (second generation). What makes mRNA vaccines stand out is their utilization of RNA, which comes in two forms. The first form is non-replicating mRNA, consisting of the antigen encoding sequence along with 5’UTR and 3’UTR. The second form is self-replicating mRNA, where the antigen encoding sequence is engineered into the RNA virus genome, allowing for self-amplification with the help of the virus’s replicase. This unique approach significantly enhances the expression of the antigen gene within cells, even with minimal injection doses.

The Advantages of mRNA Vaccines

The remarkable advantages of mRNA vaccines have propelled extensive research and widespread application in the field. One of their key strengths is the ease of preparation. The active RNA component is obtained through in vitro transcription using linear DNA as a template. During this process, the stability and translation efficiency of mRNA can be improved through codon optimization, nucleoside modification, and the use of auxiliary delivery systems. These optimizations ensure high specificity and robust stability of the nucleic acid vaccine. Additionally, the safety profile of mRNA vaccines is another major factor driving their widespread adoption. Since mRNA does not integrate into the host genome, there is no risk of infection or insertion mutation. Moreover, mRNA can be naturally degraded through normal cellular processes.

Enhancing In Vitro Transcription with Technology

At Synbio Technologies, we leverage cutting-edge technology to enhance the process of in vitro transcription. Our NGCodon^TM optimization technology simplifies the process of optimizing mRNA sequences by adjusting codon composition, GC content, and simplifying the secondary structure of mRNA. We also remove modules that hinder efficient expression, leading to improved mRNA translation efficiency and stability.

Streamlined Template Preparation

To streamline template preparation for in vitro transcription, we have developed a vector containing a poly(A) tail of over 100 base pairs. This vector facilitates fast and efficient template preparation by utilizing flat-terminal enzymes or class II restriction endonuclease enzymes (e.g., BspQI) to produce blunt or double-stranded DNA templates.

Mastering In Vitro Transcription of RNA

Synbio Technologies specializes in synthesizing various types of RNA, including sgRNA, lncRNA, and mRNA, using in vitro transcription technology. Our platform utilizes a linear DNA sequence as the template and employs T7, T3, or SP6 RNA polymerase to synthesize RNA from DNA.

Fine-tuning with Cap, Tail, and Nucleoside Modifications
A mature mRNA molecule consists of a cap structure, 5′ UTR, 3′ UTR, an open reading frame (ORF), and a poly(A) tail. The cap structure and poly(A) tail form a “closed loop” that significantly influences mRNA stability and translation efficiency. At Synbio Technologies, we offer the ability to add different modifications to RNA sequences based on our customers’ needs. These modifications further enhance the stability and translation efficiency of RNA products.

Cap Structure for Enhanced Stability and Translation Efficiency
In eukaryotes, cap structures are categorized into three types: m7GpppXpYp (Cap0), m7GpppXmpYp (Cap1), and m7GpppXmpYmp (Cap2). The cap structure is formed when the 7th carbon atom of G is methylated to form m7GpppN. Cap0 represents the basic structure, while Cap1 and Cap2 involve additional methylation at the 2′-O site of the first nucleotide and both the first and second nucleotides, respectively. Cap1 is widely found in eukaryotic mRNA and studies have shown that mRNA with Cap1 structure, modified by in vitro transcription, exhibits higher stability and translation efficiency.

Poly(A) Tail: Safeguarding mRNA Integrity
The Poly(A) tail plays a critical role in protecting mRNA from degradation. Most mRNA degradation initiates from the Poly(A) tail. By using an experimental approach, we discovered that mRNA with a 120nt Poly(A) tail demonstrated higher stability and translation efficiency compared to those with shorter tail lengths of 40nt or 80nt.

Modified Nucleosides: Fine-tuning mRNA Properties
Mammalian mRNA commonly undergoes modifications such as N1- and N6-methyladenosine (m1A, m6A, m6Am), 3- and 5-methylcytosine (m3C, m5C), 5-hydroxymethylcytosine (hm5C), pseudouridine (ψ), and 2′-O-methylation (Nm). These modifications can activate the innate immune system through toll-like receptors (TLRs). However, the introduction of modified nucleosides, such as pseudouridine (ψ), 5-methylcytidine (m5C), N6-methyladenosine (m6A), 5-methyluridine (m5U), and 2-thiouridine (s2U), in synthesized mRNA sequences can reduce the immunogenicity and improve the stability of mRNA, as they no longer activate most TLRs.

At Synbio Technologies, we continuously enhance our bio-design and technological capabilities to meet market demands and cater to the comprehensive scientific research needs of our customers. We are committed to advancing mRNA vaccines and revolutionizing immunization.