Technical answers on synthesis, chemistry, strand format and quality control.
Backbone, sugar, base and delivery chemistry address different requirements. The question is how to investigate that design space in long donors.
| Molecule | First US approval | Length | Chemistry classes and format |
|---|---|---|---|
| Fomivirsen | 1998 | 21 nt | Antisense; phosphorothioate backbone. Commercially withdrawn. |
| Mipomersen | 2013 | 20 nt | Antisense; phosphorothioate, 2′-O-methoxyethyl sugars and 5-methylcytosine. |
| Nusinersen | 2016 | 18 nt | Antisense; phosphorothioate, 2′-O-methoxyethyl sugars and methylated pyrimidines. |
| Inclisiran | 2021 | 21 / 23 nt | Duplex siRNA, not single-stranded DNA; 2′-O-methyl, 2′-fluoro, phosphorothioate and GalNAc. |
| Tofersen | 2023 | 20 nt | Antisense; phosphorothioate, 2′-O-methoxyethyl sugars and 5-methylcytosine. |
| Eplontersen | 2023 | 20 nt | Antisense; phosphorothioate / phosphodiester backbone, 2′-O-methoxyethyl, 5-methylcytosine and GalNAc. |
| Donidalorsen | 2025 | 20 nt | Antisense; phosphorothioate / phosphodiester backbone, 2′-O-methoxyethyl, methylated pyrimidines and GalNAc. |
Lengths and structures: FDA 2025 oligonucleotide workshop, slide 6; donidalorsen prescribing information, section 11. This is a set of examples, not an exhaustive inventory.
Every molecule above has strands shorter than 40 nucleotides. Chemistry has carried the oligonucleotide field this far at short length. Moligo brings the principle of internal modification to gene-length DNA through co-development.
Combining chemistries can balance competing requirements. It does not mean every molecule uses the same number or type of modifications.
Inotersen and eplontersen share an antisense sequence. Eplontersen changes the backbone pattern and adds a GalNAc conjugate for liver delivery.
Sources: inotersen US prescribing information (2018) and eplontersen US prescribing information (2023).
Moligo fine-tunes modification density with precision. Density is set by the ratio of modified to natural bases, and by ligand conjugation to built-in clickable sites. Our ssDNA synthesis services reach up to 10 kb; modification chemistry and length are agreed within each partnership. This approach takes inspiration from the global nucleoside substitution used in modified mRNA.
Karikó et al., Immunity 2005. Nobel Prize in Physiology or Medicine 2023.
Read the donor evidence and its limitsAt 99.5% coupling efficiency, 200 coupling steps give a theoretical 36.7% full-length fraction before purification.
Each step multiplies the yield from the previous step. The model is yn, where y is the per-step yield and n is the number of coupling steps. It does not predict final purity after processing.
Closely related truncation and deletion species can be difficult to separate as length increases. Resolution depends on the analytical and purification method. A numerical purity claim needs a measurement at the delivered length.
A sequence substitution can leave strand length unchanged, so routine size purification need not separate a variant from its intended sequence.
Amplification creates a population of molecules. Variants can arise at different positions; early errors can also be propagated. Polymerase identity, reaction history and template quality therefore matter.
Size and purity assays answer different questions from sequence-resolved measurements. Ask for a product-level variant assessment rather than using an enzyme fidelity number as the final product specification.
Strand-displacement reactions can also form chimeras through displaced-end re-priming. The published MDA mechanism is evidence for a risk to assess, not a universal rearrangement rate for every amplification process.
An approximately 17.1-fold difference in cGAS binding affinity was measured for duplex versus single-stranded DNA in one purified-protein assay.
Kranzusch et al., Cell Reports 2013;3:1362. Kd: 87.6 nM for dsDNA and approximately 1.5 µM for ssDNA; N-terminally truncated cGAS. Binding is not activation.
Residual dsDNA is complementary or duplex material present in a preparation intended to be single-stranded. Its fraction and size distribution can affect biological interpretation.
Length-dependent signalling varies with DNA concentration and cellular context. A 45 bp cutoff is not a universal biological threshold or a substitute for a release specification.
Luecke et al., EMBO Reports 2017;18:1707.
Ask which assay measures the duplex fraction, its detection limit and how the residual material’s size is characterised. A gel image without assay validation does not establish a numerical detection limit.
Two 25-base adaptors span 1% of a 5,000-base donor; one 100-base annealed patch spans 2%, by length arithmetic.
Those fractions describe how far the added component reaches. They do not establish its substitution fraction or biological activity.
Enzymatic incorporation can distribute chemistry throughout the molecule in the selected base channels. Moligo sets the modified fraction in the reaction pool, and tunes density by the ratio of modified to natural bases. We do not specify individual positions or regions.
Internal modification gave 41 ± 15% HDR versus 24 ± 6% for end-modified and 15 ± 5% for unmodified short donors, with the DNA-PK inhibitor AZD-7648.
Kanke et al., Nucleic Acids Research 2024;52:13561, Figure 1C. CFTR editing in airway basal cells, 800 nM donor.
No monotonic benefit was established. In that experiment, modification every third base gave 11 ± 15% HDR, also with AZD-7648. More modification was not better under those conditions.
The studied donor used defined spacing. Moligo’s long ssDNA uses tunable incorporation, set by the ratio of modified to natural bases. A matched kilobase-length comparison is still needed; the short-donor result cannot be presented as Moligo product performance.
For a 0.6 kb donor in human HSPCs, circular ssDNA gave 45.2 ± 5.0% knock-in versus 8.8 ± 3.6% for linear ssDNA under the tested conditions.
Letort et al., Nature Communications 2025;16:10125. TALEN editing with viability and HDR enhancers.
Topology and chemical modification are different variables. This comparison supports investigation of circular format in the tested workflow. It does not demonstrate a benefit from chemical modification.
Choose format through matched testing in the target cell type and editing protocol. Track viability, recovery and usable edited-cell yield as well as knock-in percentage.
Competing interests: Moligo employees are co-authors and hold equity. Cellectis authors also disclose employment and equity interests. See the paper for the full statement.
The 18 to 23 nucleotide examples on our chemistry page illustrate short therapeutic designs, not a universal 40-base limit.
FDA 2025 oligonucleotide workshop, slide 6.
Length reflects mechanism, delivery, sequence recognition and manufacturability. Chemical synthesis alone does not explain the length of every approved medicine.
Backbone, sugar and delivery modifications can address different requirements within one molecule. Inclisiran is a duplex siRNA, so it should not be described as a single-stranded DNA medicine.
The relevant question for long DNA donors is which chemistries can be incorporated and remain compatible with repair. That needs donor-specific evidence.
Your product was discontinued, or your supplier cannot hold the specification. Here is what we need from you, what we commit to, and how we prove the new material behaves like the old.
Start with the original sequence and end structure, including any added bases. Share the required quantity, concentration, buffer, application and acceptance criteria.
Agree identity, full-length content, topology and residual dsDNA measurements. Then define a bridging experiment using the existing workflow as a comparator where material remains available.
Moligo provides linear and circular ssDNA services. Chemically modified long ssDNA is a separate co-development programme for selected partners.
| Production route | Error behaviour | How the single strand is obtained | What has to be specified |
|---|---|---|---|
| Thermocycled amplification | Polymerase errors can produce a heterogeneous variant population. Routine size purification does not establish sequence correctness. | Process-dependent: for example, strand separation after duplex amplification or asymmetric amplification. | Polymerase, effective doublings, product variant burden, residual dsDNA and assay sensitivity. |
| Strand-displacement amplification | Displaced 3′ ends can re-prime elsewhere. This chimera mechanism was characterised in an MDA workflow; it is not a universal rate for RCA. | Depends on the reaction architecture and downstream processing. | Rearrangement assessment with a sequence-resolved method, such as long-read sequencing; residual dsDNA. |
| Strand separation from duplex | Inherits upstream sequence variants; separation is not sequence verification. | For example, exonuclease digestion or affinity capture. Residual complementary material must be measured. | Residual dsDNA fraction, its size distribution, assay and limit of detection. |
| Bacterial or phage production | Host and construct selection affect sequence stability and compatibility. | Production and recovery from the biological system. | Sequence identity, endotoxin, host DNA and RNA carryover. |
| Solid-phase chemical synthesis | Coupling losses compound. At 99.5% per step, 200 steps give a theoretical 36.7% full-length fraction before purification. | Single-stranded synthesis, followed by cleavage, deprotection and purification. | Full-length content at the delivered length, truncation profile and analytical method. |
| Moligo enzymatic synthesis | Isothermal rolling-circle amplification from a sequence-verified production template. Product characterisation is agreed per project. | Agreed per project. | Incorporated chemistry and fraction, strand format, full-length content, residual dsDNA and measurement methods. |
Chimera mechanism: Lasken and Stockwell, BMC Biotechnology 2007;7:19. Coupling arithmetic: 0.995200 = 36.7%. This is a theoretical pre-purification model, not a delivered-purity specification.
In a purified-protein assay, cGAS bound dsDNA with a Kd of 87.6 nM, versus approximately 1.5 µM for ssDNA. The ratio is about 17.1. These are binding constants, not activation thresholds.
Kranzusch et al., Cell Reports 2013;3:1362. Fluorescence-anisotropy assay using labelled oligonucleotides and a cGAS construct lacking its N-terminal 150 amino acids.
Activation also depends on DNA length, concentration and cellular context. Experiments with 88 to 4,003 bp duplexes found stronger responses to longer DNA at low doses. A universal 45 bp activation threshold is not supported by that study.
Luecke et al., EMBO Reports 2017;18:1707. Binding affinity alone does not predict a type I interferon response.
Ask for the residual dsDNA fraction and its size distribution, measured with an identified assay and detection limit. A percentage alone does not describe the impurity. Moligo’s project-specific limits and additional assays are agreed before work begins.
Compare analytical requirements| Ask | Why it matters | What an incomplete answer looks like | Moligo’s answer |
|---|---|---|---|
| Which polymerase and how many effective doublings? | Process history affects variant burden. Routine size purification does not certify sequence identity. | An enzyme fidelity figure offered as a finished-product variant rate. | Isothermal rolling-circle amplification from a sequence-verified template. Product-level variant assessment and process details: agreed per project. |
| Was ssDNA produced directly or recovered from duplex material? | The route determines which intermediates and carryover need measuring. | “Not detectable” without an assay or detection limit. | Strand-production and recovery details: agreed per project. |
| How much residual dsDNA, of what size, measured how? | Duplex content and size can affect biological interpretation. Assay sensitivity must be stated. | A gel image without a validated limit of detection. | Published service QC includes gel and nuclease testing. Numerical residual dsDNA limits and size-distribution methods: agreed per project. |
| Where does the chemistry sit? | Two 25-base adaptors span 1% of a 5,000-base donor by length arithmetic. | “Modified DNA” without location, chemistry or fraction. | Global substitution with per-base-channel dose control. Incorporation is tunable. Product composition and feasibility: agreed per project. |
| Is circular ssDNA available, and how is format purity measured? | Topology is a separate design variable from chemical composition. | A purity percentage without an assay. | Linear and circular ssDNA services. Published circular-format checks include S1 and exonuclease challenges. Quantitative format-purity specification: agreed per project. |
Adaptor arithmetic assumes two 25-base adaptors on a 5,000-base donor. Analytical requirements vary by construct and use. See Moligo’s published service QC criteria.
