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Construction and Gene Expression Analysis of a Single-Stranded DNA Minivector Based on an Inverted Terminal Repeat of Adeno-Associated Virus

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Abstract

The plasmid vectors currently used for nonviral gene transfer have the disadvantage of carrying a bacterial backbone and an antibiotic resistance gene, which may cause side effects. The adeno-associated virus (AAV) genome is a linear single-stranded DNA (ssDNA) molecule with palindromic inverted terminal repeat (ITR) sequences forming double-stranded DNA (dsDNA) hairpin (HP) structures at each end. Based on the AAV genome, we constructed an AAV-ITR ssDNA minivector that consists of a GFP expression cassette flanked by both ITR sequences of 125 nucleotides. The minivectors were produced by digestion of the parental plasmids followed by denaturation. The self-complementary inverted T-shaped HP structure of the minivector was automatically formed. The HEK 293T cells were transfected with the AAV-ITR ssDNA minivector, plasmid, and dsDNA expression cassette. The results showed that AAV-ITR ssDNA minivector had relatively low gene expression efficiency in vitro. However, we found that the GFP expression efficiency of the D sequence-deleted AAV-ITR ssDNA minivector was significantly increased and was similar to those obtained with the plasmid and dsDNA expression cassette. Our data suggest that the AAV-ITR ssDNA minivector may be a new type of gene expression vector for gene therapy besides the virus and plasmid.

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Abbreviations

AAV:

Adeno-associated virus

ITR:

Inverted terminal repeat

HP:

Hairpin

ssDNA:

Single-stranded DNA

dsDNA:

Double-stranded DNA

pUC57-minivector:

The plasmid consists of the double-stranded DNA expression cassette flanked by two AAV inverted terminal repeats (ITRs)

pUC57-minivector-EGFP:

The plasmid consists of the GFP expression cassette flanked by two AAV inverted terminal repeats (ITRs) (as the parent plasmid)

ds-minivector-EGFP:

The double-stranded GFP expression cassette

AAV-ITR ssDNA minivector:

The single-strand minivector consists of the DNA expression cassette and two palindromic ITR sequences

AAV-ITR-3′ m ssDNA minivector:

The minivector with a 3′-end mutation

AAV-D△-ITR ssDNA minivector:

D sequence-deleted AAV-ITR ssDNA minivector

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Acknowledgments

This work was supported by the National Natural Science Foundation of China (Grant No. 81371670 and 3097880), the Natural Science Foundation of Jiangsu Province (Grant No. BK2012629).

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Authors and Affiliations

  1. Key Lab of Bio-Medical Diagnostics, CAS, Institute of Biomedical Engineering and Technology, CAS, No. 88 Keling Road, Suzhou New District, Suzhou, 215163, People’s Republic of China

    Han Ping, Xiaomei Liu, Dongqin Zhu & Chun Zhang

  2. School of Life Science and Technology, China Pharmaceutical University, Nanjing, 211198, People’s Republic of China

    Han Ping & Taiming Li

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  1. Han Ping
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  2. Xiaomei Liu
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Correspondence to Chun Zhang.

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Han Ping and Xiaomei Liu have contributed equally as first authors.

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Ping, H., Liu, X., Zhu, D. et al. Construction and Gene Expression Analysis of a Single-Stranded DNA Minivector Based on an Inverted Terminal Repeat of Adeno-Associated Virus. Mol Biotechnol 57, 382–390 (2015). https://doi.org/10.1007/s12033-014-9832-3

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