A method for assisted sorting of liposomes using single-stranded DNA nanostructures
A nanostructure and liposome technology, which is applied in the directions of liposome delivery, pharmaceutical formulations, and medical preparations of inactive ingredients, etc., can solve problems such as uneven products, and achieve improved uptake rate, good stability, and cost. high effect
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Embodiment 1
[0041] Example 1 Sequence design of single-stranded DNA nanostructures
[0042] According to the design principle of the PX structure, taking a triangle as an example, two parallel double helices in each side are cross-connected to the other strand at the points close to each other. This structure can be assembled by a single-stranded DNA. The length of the PX is designed to be between 10 and 20 nm on each side so that its morphology can be clearly seen under an atomic force microscope. Three PX molecules were then covalently linked head-to-tail through 5'-3' to 5'-3' linkages to form triangles to ensure that the nanostructures to be generated could self-assemble from long ssDNA of about 520 nt in length (e.g. figure 1 ), whose sequence is shown in the sequence of SEQ ID NO: 1. Several TN (N=5 or 7) loops were placed at some tips to relax constraints in the edge-edge connection process.
Embodiment 2
[0043] Example 2 Construction of Phagemids Containing Complete DNA Nanostructure Sequences and Deoxyribozyme Cleavage Substrates
[0044] The complete sequence inserted into the phagemid vector consists of two parts: the deoxyribose recognition site and the nanostructure sequence site. The nanostructure sequence is in the middle, flanked by DNAzyme recognition sites. About 10 to 20 nucleotides were randomly selected as spacer sequences and distributed between the sites of deoxyribozymes and DNA nanostructures to avoid steric hindrance between the two, thus ensuring that the former was amplified in ssDNA The hairpin structure is normally formed to cut normally even if there is a large structure nearby. Taking the triangular structure as an example, a highly active self-cleaving DNAzyme (I-R1a) with a length of 45 nt (sequence: 5'-GATGTACAGCCATAGTTGAGCATTAAGTTGA / AGTGGCTGTACATC-3') is buried on both sides of the nanostructured site. When DNA becomes single-stranded, I-R1a forms...
Embodiment 3
[0045] Example 3 Helper phage-assisted amplification of single-stranded DNA
[0046] A single colony transformed with p3024-triangle was grown overnight at 37°C in 20 ml of LB medium containing ampicillin (100 μg / mL). 300mL volume of 2x YT medium (16.0g / L trypsin, 10.0g / L yeast extract, 5.0g / L NaCl, 5mM MgCl 2 ) were inoculated with 3 mL of overnight cultures and shaken at 250 r / min. When the OD600 of the culture reached about 0.4-0.5, VCSM13 helper phage was added to it to an MOI of 20. After 30 minutes, kanamycin (final concentration 50 μg / mL) was added to the culture to select for infected cells. After about 4.5 hours, the cultures were collected and centrifuged at 4000 rcf for 15 minutes at 4°C to remove the cells. The supernatant was transferred to a clean bottle and dissolved with PEG 8000 (40 g / L) and NaCl (30 g / L). The mixture was incubated on ice for 30 minutes and then centrifuged at 5000 rcf for 30 minutes at 4°C. The supernatant was discarded and the phagemid p...
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