STEP 1
Paste the target DNA or the actual amplicon. Plain sequence or single-record FASTA; A/T/C/G only, 36–1000 nt.
Argonaute detection design
gDNA Designer
Online guide DNA design for Argonaute-based detection such as PfAgo and TtAgo. Paste a target sequence and get the complete guide set in one go — gDNA1, gDNA2, gDNA3 and Reporter DNA — with cut sites, scores and a schematic side by side; check the design, then order. Calculations currently follow PfAgo rules.
What is gDNA Designer
gDNA Designer is EZassay’s free online Argonaute guide DNA design tool, built for DNA-guided detection systems such as PfAgo and TtAgo. Paste a target DNA or amplicon, and one submission returns a complete, ready-to-synthesize detection set: gDNA1, gDNA2, gDNA3 and Reporter DNA. Calculations currently follow PfAgo rules.
An Argonaute assay such as PfAgo relies on a cascade, not a single guide: gDNA1 and gDNA2 cut the same strand at two sites, and the fragment between them is gDNA4; gDNA3 cuts within that interval on the opposite strand; Reporter DNA is the reverse complement of gDNA4 and produces the readable signal. The four sequences constrain one another through their cut coordinates — change one and the others have to follow.
Designing that by hand is easy to get wrong: every guide must satisfy length, GC, homopolymer and palindrome constraints at the same time, while the three cut sites must keep the right relative positions. gDNA Designer builds those rules into the engine, returns complete cascades from a single submission, ranks them by overall cascade score, and never lets a failing candidate into the results.
Every cascade lists binding interval, binding strand and cut position for each sequence, and a schematic draws the duplex, hybridization sites and all three cuts together. Copy a whole set or export CSV — gDNA sequences carry the 5′-P- phosphorylation prefix automatically — and check the design on the page before deciding which set to order.
Complete set
gDNA1 / gDNA2 / gDNA3 / Reporter DNA returned together — no manual assembly.
Cuts you can see
The schematic shows the duplex, hybridization sites and three cut sites; every sequence lists its interval and strand.
Rules built in
PfAgo length preferences plus GC, homopolymer, palindrome, end-complementarity and primer-avoidance constraints enforced by the engine.
Ranked as cascades
Scored as whole cascades rather than single guides — start validating from the top of the list.
How to use it
STEP 2
Confirm the enzyme. PfAgo and TtAgo both use DNA guides, but the engine currently implements PfAgo rules only, so the page always calculates for PfAgo and there is nothing to choose; TtAgo is not supported.
STEP 3
If you already have amplification primers, enter them. Primers are not design targets: candidates overlapping a primer region are discarded, and candidates with local complementarity to a primer lose points.
STEP 4
Set how many cascades to return (1–30, defaults to 20 here), then review them on the results page and check the cut relationships against the schematic.
Parameters and rules
Input parameters
Target sequence
Required. Target DNA or amplicon, A/T/C/G only, 36–1000 nt. Plain sequence or single-record FASTA (header lines are ignored).
Enzyme
Argonaute preset. Only PfAgo (PFAGO) is supported today and the page submits it automatically; TtAgo and other values are rejected by the service.
Forward / reverse primer
Optional; not a design target. The engine locates each primer and its reverse complement on both strands: candidates overlapping those regions are discarded, and a guide sharing any ≥8 nt stretch with a primer loses 8 points.
Result count
1–30, defaults to 20 on this page. Cascades are returned by descending total score.
Filtering rules
Length
Tried in tiers, 16 → 17 → 18 → 15 nt: 16-mers first, others only if there are not enough cascades.
GC content
40%–80%; anything outside is discarded.
Homopolymers
No GGGG, and no run of five or more A, C or T.
Palindromes and ends
Self-reverse-complementary guides are discarded, as are guides whose first and last 5 nt are reverse-complementary; that last rule is relaxed only when nothing else survives.
Scope
All of the above apply equally to gDNA1–4 — gDNA4 is never synthesized, but it still has to pass.
Cascade relationships
gDNA1 / gDNA2
Cut the same strand at the start and the end of the gDNA4 interval; the fragment released between those two cuts is gDNA4.
gDNA3
Comes from the opposite strand; its cut must land on position 1 or 2 at the 5′ end of gDNA4 as mapped onto that strand. A gDNA3 that exactly mirrors gDNA1 on the opposite strand is excluded.
Cut position
Fixed at the boundary 10 nt before the end of the binding window — between guide positions 10 and 11 counting from the 5′ end, the canonical Argonaute cleavage site.
Reporter DNA
The reverse complement of gDNA4; it produces the readable signal.
Coordinates
The API returns 0-based half-open intervals; the page shows 1-based inclusive positions and re-bases reverse-strand coordinates onto the forward strand.
Scoring rules
Six dimensions
Each sequence gets a 0–100 score from six weighted dimensions: binding ΔG (35%), secondary-structure MFE (30%), GC (15%), homopolymer (10%), terminal base (5%) and Tm (5%).
Thermodynamic conditions
ΔG and Tm are estimated with a nearest-neighbor model at 37 °C, 150 mM Na⁺ and 200 nM strand concentration; MFE uses ViennaRNA when available, falling back to a local approximation otherwise.
Terminal base and homopolymers
A leading A or T scores 100, a leading C or G scores 40; a longest run of ≤2 identical bases scores 100, a run of 3 scores 70.
Cascade total
The mean of the four per-sequence scores (gDNA1, gDNA2, gDNA3, gDNA4). Ties go to the cascade with fewer warnings.
Diversity sampling
Not simply the top N: a high-scoring pool is grouped by cut-site skeleton, sampled for diversity, then sorted by total score — so the list is not filled with near-duplicates that differ by a single base.
Input parameters
Target sequence
Required. Target DNA or amplicon, A/T/C/G only, 36–1000 nt. Plain sequence or single-record FASTA (header lines are ignored).
Enzyme
Argonaute preset. Only PfAgo (PFAGO) is supported today and the page submits it automatically; TtAgo and other values are rejected by the service.
Forward / reverse primer
Optional; not a design target. The engine locates each primer and its reverse complement on both strands: candidates overlapping those regions are discarded, and a guide sharing any ≥8 nt stretch with a primer loses 8 points.
Result count
1–30, defaults to 20 on this page. Cascades are returned by descending total score.
Scoring rules
Six dimensions
Each sequence gets a 0–100 score from six weighted dimensions: binding ΔG (35%), secondary-structure MFE (30%), GC (15%), homopolymer (10%), terminal base (5%) and Tm (5%).
Thermodynamic conditions
ΔG and Tm are estimated with a nearest-neighbor model at 37 °C, 150 mM Na⁺ and 200 nM strand concentration; MFE uses ViennaRNA when available, falling back to a local approximation otherwise.
Terminal base and homopolymers
A leading A or T scores 100, a leading C or G scores 40; a longest run of ≤2 identical bases scores 100, a run of 3 scores 70.
Cascade total
The mean of the four per-sequence scores (gDNA1, gDNA2, gDNA3, gDNA4). Ties go to the cascade with fewer warnings.
Diversity sampling
Not simply the top N: a high-scoring pool is grouped by cut-site skeleton, sampled for diversity, then sorted by total score — so the list is not filled with near-duplicates that differ by a single base.
Filtering rules
Length
Tried in tiers, 16 → 17 → 18 → 15 nt: 16-mers first, others only if there are not enough cascades.
GC content
40%–80%; anything outside is discarded.
Homopolymers
No GGGG, and no run of five or more A, C or T.
Palindromes and ends
Self-reverse-complementary guides are discarded, as are guides whose first and last 5 nt are reverse-complementary; that last rule is relaxed only when nothing else survives.
Scope
All of the above apply equally to gDNA1–4 — gDNA4 is never synthesized, but it still has to pass.
Cascade relationships
gDNA1 / gDNA2
Cut the same strand at the start and the end of the gDNA4 interval; the fragment released between those two cuts is gDNA4.
gDNA3
Comes from the opposite strand; its cut must land on position 1 or 2 at the 5′ end of gDNA4 as mapped onto that strand. A gDNA3 that exactly mirrors gDNA1 on the opposite strand is excluded.
Cut position
Fixed at the boundary 10 nt before the end of the binding window — between guide positions 10 and 11 counting from the 5′ end, the canonical Argonaute cleavage site.
Reporter DNA
The reverse complement of gDNA4; it produces the readable signal.
Coordinates
The API returns 0-based half-open intervals; the page shows 1-based inclusive positions and re-bases reverse-strand coordinates onto the forward strand.
How the design works
STEP 1
Normalize the input
01Strip FASTA headers and all whitespace, then uppercase the sequence.
02Validate alphabet and length: A/T/C/G only, 36–1000 nt.
03If primers were supplied, mark the regions they cover so candidates can avoid them.
STEP 2
Build the candidate pool
01Using the PfAgo preset, slide a window along both the forward and reverse strands, taking 16-mers first and opening up 17, 18 and 15 nt only when there are not enough cascades.
02Hard-filter every candidate: GC range, GGGG runs, long A/C/T homopolymers, self-palindromes, 5-nt end complementarity and primer-region overlap.
03Record each candidate’s binding interval, strand and cut position (10 nt before the end of the binding window), then index candidates by cut site — cascade assembly depends entirely on those coordinates.
STEP 3
Assemble cascades
01Enumerate gDNA4 intervals on each strand (16–18 nt, 15 as a fallback); gDNA4 itself must pass the same filters and scoring.
02On the same strand, find guides whose cut lands exactly on the start and on the end of that interval — the top 3 by score at each boundary become the gDNA1 and gDNA2 candidates.
03On the opposite strand, find guides that cut at position 1 or 2 from the 5′ end of gDNA4 to serve as gDNA3, again keeping the top 3; each gDNA4 yields at most 5 cascades.
STEP 4
Score and rank
01Take the reverse complement of gDNA4 as Reporter DNA — all four deliverable sequences are now fixed.
02Score every sequence on six weighted dimensions — ΔG, MFE, GC, homopolymer, terminal base and Tm; the cascade total is the mean of the four per-sequence scores for gDNA1–4.
03Instead of taking the top N outright, draw from a high-scoring pool grouped by cut-site skeleton for diversity, then return cascades by descending total score, fewer warnings first on ties.
Frequently asked questions
What is PfAgo?
PfAgo (Pyrococcus furiosus Argonaute) is an Argonaute protein from a hyperthermophilic archaeon. Guided by a short single-stranded DNA, it finds its target by base complementarity and cleaves it, which makes it a common workhorse for isothermal nucleic acid detection. This tool currently supports PfAgo only.
What is TtAgo, and how does it differ from PfAgo?
TtAgo (Thermus thermophilus Argonaute) is an Argonaute protein from a thermophilic bacterium. Like PfAgo it cleaves target DNA using a short 5′-phosphorylated DNA guide, but its origin, working temperature and guide preferences differ, so PfAgo design rules cannot simply be reused. This tool currently implements PfAgo rules only and does not support TtAgo.
What is a gDNA (guide DNA)?
A gDNA is the short single-stranded DNA loaded into the Argonaute protein. It hybridizes to the target strand by base complementarity and determines where cleavage occurs. gDNA1, gDNA2 and gDNA3 in the results are all guides you synthesize — typically 15–18 nt, and the 5′ end must be phosphorylated.
Why are three guides needed?
A PfAgo assay depends on a precisely excised target fragment. gDNA1 and gDNA2 create two cut sites on one strand, defining gDNA4; gDNA3 then cuts within that interval on the opposite strand. Remove any one of them and the cascade cannot complete.
Do I need to synthesize gDNA4?
No. gDNA4 is the fragment released by cleavage, not a deliverable. It exists to explain where Reporter DNA comes from — Reporter DNA is its reverse complement. What you order is gDNA1, gDNA2, gDNA3 and Reporter DNA.
What is Reporter DNA?
Reporter DNA is the reverse complement of gDNA4 and produces the readable signal in the assay. It must be synthesized, but without a 5′ phosphate. Its sequence is determined by the gDNA1 and gDNA2 cut positions, so whenever the guides change, Reporter DNA changes with them.
What do “cut position” and “cascade” mean?
The cut position is where Argonaute cleaves the target strand; the page shows it as a 1-based inclusive coordinate. A cascade is the complete design formed by three guides constrained through their cut positions — each “cascade” row in the results is one such set, and scoring and ranking apply to the whole set rather than to individual guides.
Should I paste the amplicon or the original gene sequence?
Paste the actual amplicon. All coordinates are relative to the sequence you submit, so using the amplicon keeps the guides inside a region that really exists in your reaction and makes primer avoidance meaningful.
What do the primer fields do?
Primers are not designed. When supplied, the engine locates each primer and its reverse complement on both strands: candidates overlapping those regions are discarded, and a guide sharing any stretch of 8 nt or more with a primer loses 8 points. This keeps guides and primers from interfering with each other in the reaction.
Why does a target sometimes return no cascades?
Usually the region is too short or its base composition is too extreme. GC must fall within 40%–80%, GGGG runs and long A/C/T homopolymers are rejected, a guide may not be self-palindromic, and three guides with correctly placed cut sites must exist on both strands at the same time. Try a different region, widen the submitted sequence, or check whether the primers cover most of it.
Why do two runs on the same sequence differ slightly?
The returned cascades are not simply the top N. A high-scoring pool is grouped by cut-site skeleton and sampled for diversity, then sorted by total score. By default the sampling seed is derived from the inputs and the current date, so the same input is stable within a day but may return a similar-but-different set the next day. Keep the task link to reproduce a given run.
How do I read the coordinates in the results?
The API returns 0-based half-open intervals; the page converts them to 1-based inclusive positions. Coordinates given in reverse-strand numbering — typically gDNA3’s — are re-based onto the forward strand, so all four intervals can be compared directly.
Related tools
Within the same assay, primer design sits upstream of Argonaute (PfAgo / TtAgo) detection; CRISPR crRNA design is an alternative technology worth comparing.
Primer & Probe Design for RPA
Online primer and probe design for isothermal amplification (RPA/RAA). A PfAgo assay usually amplifies first, and that amplicon is exactly what you paste into this page.
crRNA Design - Cas12a(Cpf1)
Online crRNA design for CRISPR/Cas12a — a different nucleic acid detection route from Argonaute, useful to compare when choosing a system.
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