# US 2025/0084407 A1 · Topogami and method for making interlocked single stranded dna rings

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- Number: US 2025/0084407 A1
- Document ID: US20250084407A1
- Status: Published application
- Kind: A1
- Application number: 18/690,806
- Filed: 2022-09-09
- Published: 2025-03-13
- Earliest claimed: 2021-09-09
- Assignees: Uniwersytet Jagiellonski
- Applicants: Uniwersytet Jagiellonski
- Inventors: Jonathan HEDDLE; Yusuke SAKAI; Gerrit WILKENS
- Web page: https://patentgrep.com/patent/US20250084407A1

## CPC

- C12N 15/11: DNA or RNA fragments; Modified forms thereof (DNA or RNA not used in recombinant technology, C07H21/00); {Non-coding nucleic acids having a biological activity}
- C12N 15/10: Processes for the isolation, preparation or purification of DNA or RNA (chemical preparation of DNA or RNA C07H21/00; preparation of non-structural polynucleotides from microorganisms or with enzymes C12P19/34)
- C12Q 1/68: involving nucleic acids
- B82Y 5/00: Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
- C12N 2310/532: Closed or circular
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- B82Y: SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES

## Abstract

It has been disclosed a method for the construction of single stranded DNA catenanes at the kilobase length scale, which can be used for the assembling scaffolds of covalently connected DNA origamis.

## Claims

### Claim 1

A nucleic acid nanostructure (topogami) characterised in that it is artificial nucleic acid nanostructure consisting of at least 2 interlocked single stranded polynucleotide rings and associated complementary staples strands capable of folding into arbitrary 2D or 3D structures, wherein each interlocked single stranded polynucleotide ring consists of more than 300 nucleotides.

### Claim 2 (depends on 1)

The topogami according to claim 1 wherein the interlocked nucleic acid sequences are DNA (“DNA Topogami”)

### Claim 3 (depends on 1)

The topogami according to claim 1 wherein the interlocked nucleic acid sequences are RNA (“RNA Topogami”)

### Claim 4 (depends on 1)

The topogami according to claim 1 where the ssDNAs is originating from M13 phage genomic DNA.

### Claim 5 (depends on 1)

The topogami according to claim 1 wherein the staple strands are RNA or DNA.

### Claim 6 (depends on 1)

The topogami according to claim 1 where the folded 2D or 3D structures are DNA or RNA origami structures.

### Claim 7 (depends on 1)

The topogami according to claim 1 consisting of 2 interlocked single stranded DNA rings and associated complementary staples, wherein:
the first interlocked single stranded DNA ring comprises Seq Id. No. 1,
the second interlocked single stranded DNA ring comprises Seq Id. No. 2,
the complementary staple associated complementary to first DNA ring comprises sequence selected among of Seq. Id. No. 3-51,
the complementary staple associated complementary to second DNA ring comprises sequence selected among of Seq. Id. No. 52-122.

### Claim 8

A method for making interlocked single stranded DNA rings whereby double stranded rings comprising following steps:
a) engineering of DNA plasmid vectors to produce a vector consisting of two scaffold domains connected by parallel recognition sequences (res) in a negatively supercoiled parent plasmid and having integrated on opposite strands of the vector and having two nickase sites, one each on opposite strands of the vector;
b) processing of the plasmid obtained in step a) by Tn3 resolvase, a serine recombinase to catalyse a site-specific recombination at the res sites to form a supercoiled catenane;
c) conversion of catenated product obtained in step b) into ssDNA by Nt.BspQI nicking and subsequent exonuclease digestion, with nicked non-catenated parent plasmid removed by exonuclease digestion;
d) purification of the catenated ssDNA product by phenol:chloroform extraction and concentration by EtOH precipitation.

### Claim 9 (depends on 8)

The method according to claim 8, wherein the nickase site used in step a) is the Nt.BspQI nickase site.

### Claim 10 (depends on 8)

The method according to claim 8, wherein the exonucleases used in step c) are Exonuclease I and III.

### Claim 11 (depends on 8)

The method according to claim 8, wherein the plasmid used in step a) which consists of a plasmid based on pMA21 plasmid which contains two recognition sequences (res) in direct repeat and one NtBspQI nickase site wherein the addition of a second NtBspQI nickase site results in the plasmid named pTopoScaf suitable for processing as describes in 9c.

### Claim 12 (depends on 2)

The topogami according to claim 2 where the folded 2D or 3D structures are DNA or RNA origami structures.

### Claim 13 (depends on 3)

The topogami according to claim 3 where the folded 2D or 3D structures are DNA or RNA origami structures.

### Claim 14 (depends on 4)

The topogami according to claim 4 where the folded 2D or 3D structures are DNA or RNA origami structures.

### Claim 15 (depends on 5)

The topogami according to claim 5 where the folded 2D or 3D structures are DNA or RNA origami structures.