Scalable biotechnological production of DNA single strand molecules of defined sequence and length

A DNase and DNA sequence technology, which is applied in the field of recombinant production of DNA single-stranded molecules, can solve the problem that starting materials cannot be obtained in a large-scale manner

Active Publication Date: 2019-06-04
TECH UNIV MUNCHEN
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the required starting material (i.e. DNA oligonucleotides) is not currently available in a large-scale manner

Method used

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  • Scalable biotechnological production of DNA single strand molecules of defined sequence and length
  • Scalable biotechnological production of DNA single strand molecules of defined sequence and length
  • Scalable biotechnological production of DNA single strand molecules of defined sequence and length

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0175] Example 1 Production of DNA-based nanorods

[0176] As a prototype, DNA-based nanorods were produced from DNA material produced exclusively by the method of the present invention. The DNA-based nanorods were designed and developed using the DNA origami design approach (see Rothemund, 2006; see also U.S. Patent Application Nos. 2007 / 117109 A1 and 2012 / 251583 A1; U.S. Patent Nos. 7,842,793 B2 and 8,501,923 B2). Ten DNA double helices arranged in parallel in a crystal lattice and cross-linked by strand junctions (see Figure 4 A). For the nanorods, a 2500 base long single-stranded DNA backbone molecule (the "scaffold strand") and 21 DNA oligonucleotides each approximately 100 bases in length (the "master product strand") are required . All required construction elements are derived from phagemids specially constructed for this purpose.

[0177] 1.1 Materials and methods

[0178] -sequence:

[0179] DNAse:

[0180] P1-[SEQ ID NO.1]-P2-P3-P2'-[SEQ ID NO.2]-P1'

[018...

Embodiment 2

[0200] Example 2 Optimization of DNase sequence

[0201] To construct our phagemids, we did not simply place a constant DNase sequence between the target oligonucleotides in the same manner as we inserted restriction enzyme binding sites. The sequence of the DNase depends on the terminal sequence of the oligonucleotide to be produced. Whereas Gu et al. (Biotechniques 2013) simply used the same end sequence and thus the same DNase, our method uses a different DNase sequence to produce different target oligonucleotide sequences.

[0202] To identify essential bases in the DNase sequence, we screened about 40 different variants of each DNase as oligonucleotides (two examples see Figure 6A ) and five versions of full-length phagemids (for two examples see Figure 6B ). We found some bases that were not classified as essential in the original literature on DNase (Gu et al., JACS 2013). This allows us to construct phagemids that are fully cleaved to the desired product in an ac...

Embodiment 3

[0220] Example 3 Other nanostructures assembled from DNA oligonucleotides produced using the DNase-based method of the invention

[0221] In addition to nanorods, we designed 48-helix tubes assembled from 3200 base-long scaffolds and 31 main product oligonucleotides (see Figure 7A ). As with nanorods, all major products for this 48-helix tube are encoded on one phagemid, and the backbone of the phagemid acts as a scaffold. As with the nanorods, due to the inherently precise 1:1 stoichiometry, the objects assembled efficiently into the desired shape without using the primary product in excess of the scaffold.

[0222] To demonstrate the applicability of our method to the assembly of existing full-scale DNA origami objects, we generated all 161 Phagemids seeded with primary product oligonucleotides (see Figure 7B ). In contrast to nanorods and 48-helix bundles, the main product for pointers is distributed on 4 separate phagemids and uses a single scaffold (M13mp18, 7249 ba...

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Abstract

The present invention relates to a method for the recombinant production of DNA single stranded molecules, comprising the steps of (1) providing a pseudogene nucleic acid; (2) integrating the pseudogene nucleic acid into a vector, transforming bacterial cells with said vector and producing a precursor ssDNA from said vector under bacterial culture conditions; (3) isolating the precursor ssDNA fromthe bacterial culture; (4) digesting the precursor ss DNA under reaction conditions where self-cleaving DNA sequences become active; and (5) separating and obtaining the target single stranded DNA oligo- or polynucleotide(s). The method of the present invention is suitable for the mass production of DNA single stranded molecules. The present invention further relates to the use of the target single stranded DNA oligo- or polynucleotide(s), in particular in DNA nanotechnology, or as research tools.

Description

technical field [0001] The present invention relates to a method for recombinant production of DNA single-stranded molecules, the method comprising the following steps: (1) providing a pseudogene nucleic acid; (2) integrating the pseudogene nucleic acid into a vector, and transforming the pseudogene nucleic acid with the vector Bacterial cells and produce precursor ssDNA from said vector under bacterial culture conditions; (3) isolate said precursor ssDNA from said bacterial culture; (4) digest under reaction conditions that self-cleaving DNA sequences become active the precursor ssDNA; and (5) isolating and obtaining the target single-stranded DNA oligonucleotide or polynucleotide. The method of the invention is suitable for large-scale production of DNA single-stranded molecules. The invention also relates to the use of said target single-stranded DNA oligonucleotides or polynucleotides, in particular in DNA nanotechnology or as a research tool. Background technique [0...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10C12N15/11C12N15/113C12N15/115
CPCC12N15/10C12P19/34C12Q2521/345C12N15/65C12Q1/6806C12Q1/6876C12N2800/101C12N15/73C12N2795/14042
Inventor 弗罗里安·普拉托里奥斯亨德里克·迪茨
Owner TECH UNIV MUNCHEN
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