Intracellular antigen labeling and manipulation by antibodies have been long-thought goals in the field of cell research and therapy. However, a central limitation for this application is that antibodies are not able to penetrate into the cytosol of living cells. Taking advantages of small sizes and unique structures of the single-domain antibodies, here, we presented a novel approach to rapidly deliver the nanobody/variable domain of heavy chain of heavy-chain antibody (VHH) into living cells via introducing its coding mRNA, which was generated by in vitro transcription. We demonstrated that actin-green fluorescent proteins (GFP) and Golgi-GFP can be recognized by the anti-GFP nanobody/VHH, vimentin can be recognized by the anti-vimentin nanobody/VHH, and histone deacetylase 6 (HDAC6) can be recognized by the anti-HDAC6 nanobody/VHH, respectively. We found that the anti-GFP nanobody expressed via in vitro-transcribed (IVT) mRNA can be detected in 3 h and degraded in 48 h after transfection, whereas the nanobody expressed via plasmid DNA, was not detected until 24 h after transfection. As a result, it is effective in delivering the nanobody through expressing the nanobody/VHH in living cells from its coding mRNA.

中文翻译：

---

通过表达体外转录的mRNA将纳米抗体/ VHHs快速递送到活细胞中。

细胞内抗原标记和抗体操纵已成为细胞研究和治疗领域的长远目标。但是，该应用的主要局限性在于抗体无法渗透到活细胞的细胞质中。利用单结构域抗体的小尺寸和独特结构，在这里，我们提出了一种新颖的方法，通过引入其编码mRNA，将重链抗体（VHH）重链的纳米抗体/可变域快速递送到活细胞中，这是通过体外转录产生的。我们证明了肌动蛋白绿色荧光蛋白（GFP）和高尔基GFP可以被抗GFP纳米抗体/ VHH识别，波形蛋白可以被抗波形蛋白纳米抗体/ VHH识别，并且组蛋白脱乙酰基酶6（HDAC6）可以被识别分别由抗HDAC6纳米抗体/ VHH组成。我们发现通过体外转录（IVT）mRNA表达的抗GFP纳米抗体可以在转染后3小时内检测到并在48小时内降解，而通过质粒DNA表​​达的纳米抗体要在转染后24小时才能检测到。结果，通过在活细胞中从其编码mRNA表达纳米抗体/ VHH，有效递送纳米抗体。