Abstract:Bacterial DNA methyltransferases offer an approach to addressable protein targeting in macromolecular assembly that permits the construction of ordered arrays of functional proteins or peptides. This approach uses the natural recognition specificity of the bacterial DNA cytosine methyltransferase to target fusion proteins to preselected sites on a DNA scaffold through the formation of a stable covalent attachment to a mechanism-based methyltransferase inhibitor. Addressable targets on DNA molecules can readily… Show more
“…It consists of a DNA scaffold, which displays three copies of the thioredoxin redox protein for targeting, and a fluorescein label used for detection in fluorescence microscopy (Figure 1). Methods used for DNA synthesis have been described [15], [16]. The methods used to assemble and analyze the ND-Trx3 (Figure 1) have been described [17], [18].…”
Histological staining of reactive stroma has been shown to be a predictor of biochemical recurrence in prostate cancer, however, molecular markers of the stromal response to prostate cancer have not yet been fully delineated. The objective of this study was to determine whether or not the stromal biomarkers detected with a thioredoxin-targeted nanodevice could be used to distinguish the stroma associated with benign prostatic hyperplasia from that associated with PCA. In this regard, we recently demonstrated that a thioredoxin-targeted nanodevice selectively binds to reactive stroma in frozen prostate tumor tissue sections. To accomplish this, random frozen prostate tissue sections from each of 35 patients who underwent resection were incubated with the nanodevice and graded for fluorescent intensity. An adjacent section from each case was stained with Hematoxylin & Eosin to confirm the diagnosis. Select cases were stained with Masson's Trichrome or immunohistochemically using antibodies to thioredoxin reductase 1, thioredoxin reductase 2 or peroxiredoxin 1. Our results demonstrate that the graded intensity of nanodevice binding to the stroma associated with PCA was significantly higher (p = 0.0127) than that of benign prostatic hyperplasia using the t-test. Immunohistochemical staining of adjacent sections in representative cases showed that none of the two commonly studied thioredoxin interacting protein partners mirrored the fluorescence pattern seen with the nanodevice. However, thioredoxin reductase 2 protein was clearly shown to be a biomarker of prostate cancer-associated reactive stroma whose presence distinguishes the stroma associated with benign prostatic hyperplasia from that associated with prostate cancer. We conclude that the signal detected by the nanodevice, in contrast to individual targets detected with antibodies used in this study, originates from multiple thioredoxin interacting protein partners that distinguish the M2 neutrophil and macrophage associated inflammatory response in prostate cancer-associated stroma from the CD4+ T-Lymphocyte linked inflammation in benign prostatic hyperplasia.
“…It consists of a DNA scaffold, which displays three copies of the thioredoxin redox protein for targeting, and a fluorescein label used for detection in fluorescence microscopy (Figure 1). Methods used for DNA synthesis have been described [15], [16]. The methods used to assemble and analyze the ND-Trx3 (Figure 1) have been described [17], [18].…”
Histological staining of reactive stroma has been shown to be a predictor of biochemical recurrence in prostate cancer, however, molecular markers of the stromal response to prostate cancer have not yet been fully delineated. The objective of this study was to determine whether or not the stromal biomarkers detected with a thioredoxin-targeted nanodevice could be used to distinguish the stroma associated with benign prostatic hyperplasia from that associated with PCA. In this regard, we recently demonstrated that a thioredoxin-targeted nanodevice selectively binds to reactive stroma in frozen prostate tumor tissue sections. To accomplish this, random frozen prostate tissue sections from each of 35 patients who underwent resection were incubated with the nanodevice and graded for fluorescent intensity. An adjacent section from each case was stained with Hematoxylin & Eosin to confirm the diagnosis. Select cases were stained with Masson's Trichrome or immunohistochemically using antibodies to thioredoxin reductase 1, thioredoxin reductase 2 or peroxiredoxin 1. Our results demonstrate that the graded intensity of nanodevice binding to the stroma associated with PCA was significantly higher (p = 0.0127) than that of benign prostatic hyperplasia using the t-test. Immunohistochemical staining of adjacent sections in representative cases showed that none of the two commonly studied thioredoxin interacting protein partners mirrored the fluorescence pattern seen with the nanodevice. However, thioredoxin reductase 2 protein was clearly shown to be a biomarker of prostate cancer-associated reactive stroma whose presence distinguishes the stroma associated with benign prostatic hyperplasia from that associated with prostate cancer. We conclude that the signal detected by the nanodevice, in contrast to individual targets detected with antibodies used in this study, originates from multiple thioredoxin interacting protein partners that distinguish the M2 neutrophil and macrophage associated inflammatory response in prostate cancer-associated stroma from the CD4+ T-Lymphocyte linked inflammation in benign prostatic hyperplasia.
“…For example, the WRN helicase has been shown to resolve quadruplex DNA [ 15 ] and deficiency appears to result in the accumulation of non-B structures [ 10 ]. The evidence suggests that the DNMTs remain bound to non-B DNA sequences containing mispaired cytosines [ 107 ], oxidized bases [ 121 ] or DNA containing base analogs, such as deoxyuridine (dU) [ 122 ], 5azaC-dR or GuaUre-dR [ 1 ]. It follows, that in cases of helicase deficiency, DNMT sequestration at a site of hypermethylation will result in global hypomethylation, much like the effects of 5-azacytidine (5azaC-R ) , 5azaC-dR and GuaUre-dR result in hypomethylation, since tightly bound DNMTs are unable to maintain normal methylation patterns.…”
BackgroundA remarkable correspondence exists between the cytogenetic locations of the known fragile sites and frequently reported sites of hypermethylation. The best-known features of fragile sites are sequence motifs that are prone to the spontaneous formation of a non-B DNA structure. These facts, coupled with the known enzymological specificities of DNA methyltransferase 1 (DNMT1), the ATP-dependent and actin-dependent helicases, and the ten-eleven translocation (TET) dioxygenases, suggest that these enzymes are involved in an epigenetic cycle that maintains the unmethylated state at these sites by resolving non-B structure, preventing both the sequestration of DNA methyltransferases (DNMTs) and hypermethylation in normal cells.Presentation of the hypothesisThe innate tendency of DNA sequences present at fragile sites to form non-B DNA structures results in de novo methylation of DNA at these sites that is held in check in normal cells by the action of ATP-dependent and actin-dependent helicases coupled with the action of TET dioxygenases. This constitutes a previously unrecognized epigenetic repair cycle in which spontaneously forming non-B DNA structures formed at fragile sites are methylated by DNMTs as they are removed by the action of ATP-dependent and actin-dependent helicases, with the resulting nascent methylation rendered non-transmissible by TET dioxygenases.Testing the hypothesisA strong prediction of the hypothesis is that knockdown of ATP-dependent and actin-dependent helicases will result in enhanced bisulfite sensitivity and hypermethylation at non-B structures in multiple fragile sites coupled with global hypomethylation.Implications of the hypothesisA key implication of the hypothesis is that helicases, like the lymphoid-specific helicase and alpha thalassemia/mental retardation syndrome X-linked helicase, passively promote accurate maintenance of DNA methylation by preventing the sequestration of DNMTs at sites of unrepaired non-B DNA structure. When helicase action is blocked due to mutation or downregulation of the respective genes, DNMTs stall at unrepaired non-B structures in fragile sites after methylating them and are unable to methylate other sites in the genome, resulting in hypermethylation at non-B DNA-forming sites, along with hypomethylation elsewhere.
“…It results from the normal cyclical (i.e., catalytic) chemical process in which the chemical activation of the substrate by the enzyme is followed by methyltransfer and then release of the product methylated DNA. When 5F-cytosine is substituted for cytosine at the target site or when uracil is substituted for cytosine, the cycle cannot be completed and the enzyme molecule forms a Michael adduct with cytosine, stalls at the targeted site and becomes stabily bound to the DNA at the targeted recognition site (Figure ). 1 Attack of 5-fluorodeoxycytosine by DNA (cytosine-5) methyltransferases.…”
The methyltransferase-directed addressing of fusion proteins to DNA scaffolds offers an approach to the construction of protein/nucleic acid
biostructures with potential in a variety of applications. In this report I describe the use of this technology in the design and construction of
a molecular camshaft with implications in the design of independently addressable trisubstituted components for the construction of additional
nanoscale materials and devices.
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