2LTRZFP Interacts Specifically to HIV-1 DNA without Off-Target Effects as Determined by Biolayer Interferometry.

Koollawat Chupradit,Chatchai Tayapiwatana,Kanokporn Sornsuwan,On-Anong Juntit,Weeraya Thongkum

doi:10.3390/bios11030076

Abstract

Protein and DNA interactions are crucial for many cellular processes. Biolayer Interferometry (BLI) is a label-free technology for determining kinetic biomolecular interactions with high accuracy results. In the present study, we determined the kinetic binding of a zinc finger scaffold, 2LTRZFP, which formerly constructed the interfering effect on HIV-1 integration process using BLI. The competitive Enzyme-linked immunosorbent assay (ELISA) was used to initially show the specific binding of 2LTRZFP. The percentages of inhibition were 62% and 22% in double-stranded 2LTR (ds2LTR) and irrelevant DNA (dsNeg), respectively. Consequently, the binding affinity of 2LTRZFP against ds2LTR target analyzed by BLI was 40 nM, which is stronger than the interaction of HIV-1 integrase (IN) enzyme to the 2LTR circle junction. Additionally, the 2LTRZFP did not interact with the genomic DNA extracted from SupT1 cell line. This result indicates that 2LTRZFP did not exhibit off-target effects against human genome. The knowledge obtained from this study supports the prospect of using 2LTRZFP in HIV-1 gene therapy.

Highlights

Regarding the drawbacks of antiretroviral therapy which is a current standard treatment for Human Immunodeficiency Virus-1 (HIV-1) infected patients, gene therapy is one of the approaches that has been developed as an alternative treatment towards HIV-1 infection
Several anti-HIV-1 molecules have been identified for use in HIV-1 gene therapy, including RNA-based methods [1,2], and protein-based method such as scaffold proteins and gene editing proteins, for example, designed ankyrin repeat protein, zinc finger nucleases (ZFNs) transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein
The recombinant 2LTRZFP-Green Fluorescent Protein (GFP) was expressed in E. coli strain Origami B (DE3)

Summary

Introduction

Regarding the drawbacks of antiretroviral therapy which is a current standard treatment for Human Immunodeficiency Virus-1 (HIV-1) infected patients, gene therapy is one of the approaches that has been developed as an alternative treatment towards HIV-1 infection. Several anti-HIV-1 molecules have been identified for use in HIV-1 gene therapy, including RNA-based methods [1,2], and protein-based method such as scaffold proteins and gene editing proteins, for example, designed ankyrin repeat protein, zinc finger nucleases (ZFNs) transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) [3,4,5,6,7]. A zinc finger protein (ZFP), namely 2LTRZFP, was formerly established by submitting the 18 base pairs of HIV-1 2LTR target sequence into the zinc finger tools software [8]. The designed 2LTRZFP consists of 6 contiguous ZFP motifs that have binding domains cover the range of HIV-1 2LTR target sequence. The study in human T cell lines and primary peripheral blood mononuclear cells (PBMCs) demonstrated that

Methods

Results

Conclusion