INTRODUCTION

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The Opportunity

Synthetic Biology promises to deliver the knowledge, at unprecedented rates, to protect, heal, and rejuvenate the human body.  However current methods to make synthetic DNA and messenger RNA limit the creation of error free gene libraries.  Large scale automation of DNA and RNA synthesis is needed to overcome this bottleneck.   Nanopec has pioneered proprietary platforms that can deliver the full promise of Synthetic Biology by enabling the synthesis of longer DNA and mRNA sequences with unprecedented yield and quality.

DNA synthesis occurs in all eukaryotes and prokaryotes. In humans the accurate synthesis of DNA is important to avoid mutations. Mutations could lead to diseases such as cancer. For these and other fundamental reasons, DNA biosynthesis, and the machinery involved in vivo, has been studied extensively since the 1950’s.

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DNA biosynthesis in nature occurs via the polymerase chain reaction. Gene synthesis - physically creating de novo artificial gene sequences in solution, requires the ligation of tens to thousands of individual distinct oligonucleotide chains created by phosphoramidite chemistry. These, purely chemical, means to create oligonucleotides have been in existence for over 40 years. Though specific processes can be very different, they have several important features in common.  Microplates filled with frits, plastic columns filled with glass beads.  Silicon chips and split and pool automation are by far the most accurate and yield the larger number of distinct oligonucleotides.

 

Single nucleotides can be joined to form oligonucleotides that can act as a DNA template for gene manufacture. Entire genes, consisting of thousands of nucleotides (nt) bases are now possible.  Nonetheless the process is still prone to errors and thus only relatively short sequences (<200 nt) can be synthesized before the yield, after purification, falls to single digits.

 

The need for gene  manufacturing will change significantly in the years to come.  In specialized Academic areas such as creating de novo organisms, CRISPR technology will no doubt make significant contributions to the field.  CRISPR is a family of DNA sequences found in the genomes of prokaryotic organisms such as bacteria and archaea, will change how genes are altered, edited and manipulated directly into living organisms. Cas9 enzymes together with CRISPR sequences form the basis of a technology known as CRISPR-Cas9 that can be used to edit genes within organisms. This editing process has a wide variety of applications including basic biological research, development of biotechnology products, and the treatment of diseases.  However, research in this area as well as in precision medicine, gene therapy and diagnostics will continue to be strongly dependent on chemical synthesis methods for DNA, oligonucleotide synthesis to generate high purity new genes ex-vivo or to populate gene data banks to deposit the vast amount of genetic information currently being threaten with extinction.

Other Applications:

 

An established  use of synthetic DNA  is the creation of primers and probes for PCR.  PCR works by using a relatively short (100 nt to 200 nt) single stranded DNA template. The DNA double helix unwinds during replication, exposing unpaired bases for new nucleotides to hydrogen bond to. Without the primer template PCR is unable to amplify DNA. Probes are also relatively short oligonucleotide sequences that are capped with a fluorophore and a quencher. Their sequence rarely changes in the ends, which generate a hairpin structure to keep the fluorophore from emitting unless the mid-section of the probe finds a complementary strand of DNA to bind. In this case the hairpin is unfolded and the quencher and fluorophore separated by the exonuclease activity of DNA polymerase during amplification. As a result, the solution becomes fluorescent which indicates the presence of the sought DNA sequence.

 

 The fastest growth area, however, is the emerging field of mRNA vaccines.  Due to the need for effective vaccines to manage the impact of the COVID-19 pandemic mRNA vaccines have taken a center stage in the commercial uses of synthetic oligonucleotides.  Adapting these vaccines to new SARS-Cov2 variants requires de novo synthesis of the necessary mRNA oligonucleotide sequence.  In addition, vaccines for the flu and certain cancers are now being pursued.

Our Value Proposition

Nanopec patented DNAReax ceramic chips for the synthesis of DNA and RNA with significant advantages over controlled pore glass (CPG) powder columns and silicon chips.  All three methodologies use the same well proven phosphoramidites chemistry but have significant differences for the future of DNA automation.

-CPG: Although CPG has been in use for many decades it is not suitable for large scale automation of gene synthesis.  Due to its bulky nature and microfluidics needs CPG is not suitable for the assembly of the thousands of DNA pieces (oligos) needed to assemble an entire gene.   

-Silicon chips: solve this problem by using flat, optically labeled silicon micro-chips that are automatically sorted through a handful of chemical reaction baths.  Each machine is capable of sorting up to 5,000 chips at a time, each with a different oligo.  However given the smooth flat nature of the chip’s surface, and the difficulty of initiation the reaction on silicon, the amounts of oligo produced less than 1% compared to a single CPG column. 

-DNAReax nano-porous ceramic chips deliver over 70 to 100 times the amount of high quality oligo than a comparably size silicon chip (customer data).  DNAReax is manufactured to customer’s specifications, shape and size formats, as a turn key replacement of their silicon chips without any required alterations of their existing DNA automation equipment.  DNAReax chips match the optical purity and mechanical strength of silicon chips while exceeding by more than 100 times the available surface area (inside its nano-pores) to synthesize more DNA.  Additional advantages include milder conditions for starting the synthesis process and the controlled spacing of individual DNA chains to eliminate errors caused by overcrowding on a flat silicon chip surface or on the randomly sized pores of CPG.

In a series of two white papers we summarize the various established methods of oligonucleotide synthesis and take a look at emerging technologies, their advantages and their limitations.  We outline the chemical, optical and mechanical properties of DNAReax to provide high quality oligonucleotides at unprecedented yields when compared to silicon chips. 

 

Take a minute to order your own copy of these important white papers, you will be glad you did.  Let us know if you have any questions. We are here to respond them and to provide any technical assistance your specific application may require.