Chemistry throughout the strand.

Our focus is the composition of long single-stranded DNA. We incorporate modified nucleotides during enzymatic synthesis.

Internal modification.
Compositional control.

Moligo sets the modified nucleotide fraction in the reaction pool. The polymerase incorporates those nucleotides statistically throughout the product.

One pool serves the synthesis. We do not specify individual modified positions, defined spacing or different densities in selected regions.

Global substitution is the strategy biology uses for this problem. Phages hypermodify their genomes to defeat host nucleases, and the answer is always wholesale replacement of one base rather than sparse decoration. T4 replaces 100 per cent of its cytosine with 5-hydroxymethylcytosine. Phage S-2L replaces 100 per cent of its adenine with 2-aminoadenine. PBS2 replaces 100 per cent of its thymine with uracil. Modified messenger RNA is manufactured the same way: a dose set per nucleotide channel, not a map of positions.

Chemistry selection, incorporation and the resulting product composition must be established for each project.

Terminal modification

Modification throughout the strand

Schematic only. Internal positions are illustrative, not programmable modification sites.

Why our technology

The difference is where chemistry enters the process.

Moligo introduces modified nucleotides into the synthesis reaction. Chemistry can be incorporated throughout the long strand, rather than being confined to an attached end adaptor or a short annealed segment.

For a partner, that creates an additional design variable: the composition of the whole molecule. It does not provide a map of individually addressable positions.

On a 5,000-base donor, two 25-base modified end adaptors place chemistry on about 1 per cent of the molecule. The remaining 99 per cent is unmodified.

Approach	Where chemistry sits	Share of a 5 kb donor it reaches	Length ceiling	Positional control
Modified end adaptors	The two attached ends	About 1%, for two 25-base adaptors	Set by the amplification route	Yes, within the adaptor
Annealed modified oligonucleotide	One local segment	About 2%, for a 100-base patch	Set by the template	Yes, within the patch
Solid-phase synthesis	Any position in the strand it synthesises	The whole molecule, but it cannot make a 5 kb strand	About 200 bases	Yes
Moligo enzymatic synthesis	Distributed through the strand	The full length, in every base channel used	Up to 10 kb	No. Statistical incorporation, dose set by the pool

Percentages are arithmetic on the stated adaptor and patch lengths, shown so the comparison can be recalculated for any construct. Substitute your own donor length and adaptor size. Solid-phase full-length yield falls as coupling compounds: at 99.5 per cent coupling per step, a 200-base oligonucleotide is 36.7 per cent full length, and the remainder is deletion products that are near-isosequence with the target.

Enzymatic synthesis at kilobase length.

Our in vitro, isothermal platform is based on rolling-circle amplification. It uses a production template and an enzymatic reaction to make long ssDNA. Our linear and circular ssDNA service describes lengths up to 10 kb; modification feasibility is assessed within the partnership programme.

Other amplification routes can also accept modified nucleotides. Our comparison is about a specific process and product specification, not a claim that all other enzymatic methods are limited to end modification.

Measure the product, not the promise.

Amplification, strand separation and biological production each require their own process controls. A method name alone does not establish fidelity, full-length content or absence of complementary DNA.

Four questions settle any supplier comparison. Over how many doublings was the molecule amplified, and with which polymerase? Was the single strand made single, or made double and then reduced, and what is the residual double-stranded DNA figure, by what assay, at what limit of detection? Where does the chemistry sit? Is a circular format available, and at what purity?

We agree the analytical requirements with each partner: incorporated chemistry and fraction, strand format, full-length content, residual double-stranded DNA and the methods used to measure them.

A capability to test. A partnership to test it in.

The published short-donor comparison below supports investigating internal chemistry. It does not prove that Moligo’s modified long ssDNA performs better. Our partnership programme is where synthesis, composition and matched biological controls are developed together.

Discuss a modification partnership

What the evidence establishes.

Internal modification and circular topology answer different questions. The studies below should be read separately.

Kanke et al. · Nucleic Acids Research · 2024

Internal versus terminal modification in short donors.

Donor	HDR
Internal phosphorothioate modification	41 ± 15%
End-modified	24 ± 6%
Unmodified	15 ± 5%

CFTR editing in airway basal cells, 800 nM donor, with the DNA-PK inhibitor AZD-7648. Figure 1C.

The best-performing donor in this comparison used defined spacing. Denser modification reduced HDR to 11 ± 15%. More modification was not better.

Moligo’s long ssDNA uses statistical incorporation. A matched modified-versus-unmodified comparison at kilobase length remains to be demonstrated.

Read the Kanke paper

Letort et al. · Nature Communications · 2025 · With Cellectis

Circular versus linear ssDNA in HSPCs.

For the 0.6 kb donor, circular ssDNA achieved 45.2 ± 5.0% knock-in, compared with 8.8 ± 3.6% for linear ssDNA (mean ± SD). The protocol used TALEN editing and included viability and HDR enhancers.

This study supports the circular format. It does not establish an advantage for Moligo’s modified DNA over matched unmodified DNA.

Disclosure: Moligo employees and equity holders Roger Salvatori and Cosimo Ducani are co-authors. Cellectis co-authors also report employment and equity interests.

Read the Cellectis collaboration paper

Choose chemistry for the application.

Donor persistence, sensing and repair impose different requirements. The route of administration changes which ones matter.

Ex vivo editing

The donor is introduced into cells outside the body. Plasma exposure is not part of this route. Evaluate intracellular donor survival, cell viability, repair and the number of usable edited cells.

In vivo delivery

Systemic delivery also brings circulation, clearance and formulation into the design. A chemistry selected for ex vivo editing cannot be assumed to behave the same way in vivo.

Four stages to consider

Stage	Design question	Qualification
Circulation	How do nucleases and clearance affect the donor?	Relevant to systemic delivery. Not plasma exposure in ex vivo editing.
Endosome	How do DNA chemistry and formulation affect recognition?	Delivery formulation can change the effect of a base modification.
Cytosol	Does the donor persist long enough, and what sensing does that persistence provoke?	Nuclease resistance does not imply absence of immune sensing.
Nucleus	Can the donor still be read by repair machinery?	Higher modification density may reduce repair efficiency.

Chemistry is developed within the partnership.

Backbone, base and sugar modifications present different incorporation and biological questions. A chemistry discussed in the literature is not automatically an available Moligo product. We assess the combinations, fractions and lengths that can be investigated within an agreed partnership scope.

Enzymatic Injection Molding Technology.

Moligo’s platform uses sequence-verified production templates and in vitro enzymatic reactions to produce long ssDNA. Modified nucleotides are introduced through the reaction pool.

Linear and circular formats remain part of our synthesis service. We agree the sequence, chemistry, format and analytical specification for each project.

Explore our services

What we establish together

The target sequence and length.
The modified nucleotide chemistry and reaction composition.
Product composition, strand format and release testing.
The matched controls needed to interpret biological performance.

Discuss your DNA.

Tell us about your sequence, application and the chemistry you want to investigate.

Talk to our scientists