The human MutY homolog, hMYH, is an adenine-specific DNA glycosylase that removes adenines or 2-hydroxyadenines mispaired with guanines or 8-oxoguanines. In order to prevent mutations, this activity must be directed to the newly synthesized strand and not the template strand during DNA synthesis. The subcellular localization and expression of hMYH has been studied in serum-stimulated, proliferating MRC5 cells. Using specific antibodies, we demonstrate that endogenous hMYH protein localized both to nuclei and mitochondria. hMYH in the nuclei is distinctly distributed and co-localized with BrdU at replication foci and with proliferating cell nuclear antigen (PCNA). The levels of hMYH in the nucleus increased 3- to 4-fold during progression of the cell cycle and reached maximum levels in S phase compared to early G(1). Similar results were obtained for PCNA, while there were no notable changes in expression of 8-oxoguanine glycosylase or the human MutT homolog, MTH1, throughout the cell cycle. The cell cycle-dependent expression and localization of hMYH at sites of DNA replication suggest a role for this glycosylase in immediate post-replication DNA base excision repair.

No abstract

No abstract

A phosphorothioate single-stranded DNA aptamer (thioaptamer) targeting transforming growth factor-β1 (TGF-β1) was isolated by in vitro combinatorial selection. The aptamer selection procedure was designed to modify the backbone of single-stranded DNA aptamers, where 5′ of both A and C are phosphorothioates since this provides enhanced nuclease resistance as well as higher affinity than that of a phosphate counterpart. The thioaptamer selected from a combinatorial library (5 × 10 14 sequences) binds to TGF-β1 protein with an affinity of 90 nM. In this report, sequence, predicted secondary structure, and binding affinity of the selected thioaptamer (T18_1_3) are presented. KeywordsTransforming growth factor-β1; selection; aptamer; phosphorothioate; thioaptamer; chemiluminescence; entropy Transforming growth factor-β1 (TGF-β1) is a 25 kDa homodimer composed of two 12.5 kDa subunits joined by a disulfide bond. The protein is known as a multifunctional cytokine acting both through autocrine and paracrine mechanisms. The protein suppresses the immune system and is particularly associated with immune disregulation by inhibiting the proliferation of normal T lymphocytes via down-regulation of interleukin-2-mediated proliferative signals. 1 TGF-β1 also mediates tumor-promoting effects, either through differential effects on tumor and stromal cells or through alteration in the TGF-β1 responsiveness of the tumor cells themselves. 2 Inhibition of the TGF-β signaling pathway has been proposed for cancer therapy. 3 Therefore, agents that target and antagonize TGF-β1 may serve as therapeutic or research tools by modulating the function of this protein.Combinatorial selection of oligonucleotide aptamers has been employed to develop potential therapeutic agents since its introduction. 4,5 The principle of this technique is isolation and enrichment of specific nucleic acid sequences (aptamers) that bind to target molecules from a large random combinatorial library. This approach seems especially appropriate as inhibitor Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. NIH Public Access NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript development because the protein is known to bind to heparin, a polyanionic polymer like DNA 6 The selection in this study was designed to modify every 5′ of adenosine and cytosine of single-stranded DNA (ssDNA) aptamers to phosphorothioate, since phosphorothioatesubstituted aptamers typically display an enhanced affinity for targeted molecules and increased stability in the biological milieu. 7,8 Several DNA 9,10 and RNA 11,12 ...

A phosphorothioate RNA aptamer (thioaptamer) targeting the capsid protein of Venezuelan equine encephalitis virus (VEEV) was isolated by in vitro combinatorial selection. The selected thioaptamer had a strong binding affinity ($7 nM) and high specificity for the target protein. For the binding to the protein, the overall tertiary structure of the thioaptamer is required. We introduce two theoretical methods to examine the effect of phosphorothioate modification on the enhancement of binding affinity and one experimental method to examine the nature of the multiple bands of thioaptamer in a native gel.