Carbon dots (C-dots) are often synthesized, modified, and studied as a mixture. Unfortunately, the spectroscopic and biological properties measured for such C-dots assume that there is a high degree of homogeneity in the produced sample. By means of high-resolution separation techniques, we show that "as-synthesized" C-dots exist as a relatively complex mixture and that an unprecedented reduction in such complexity can reveal fractions of C-dots with unique luminescence properties. The wavelength-dependent photoluminescence commonly assigned as an inherent property of C-dots is not present in fractionated samples. While ultraviolet-visible absorption profiles reported for C-dots are typically featureless, we have found fractions of C-dots possessing unique absorption bands, with different fractions possessing specific emission wavelengths. Furthermore, fractionated C-dots showed profound differences in emission quantum yield, allowing for brighter C-dots to be isolated from an apparent low quantum yield mixture. These more luminescent fractions of C-dots displayed improved biological compatibility and usefulness as cellular imaging probes.

RNA is an important therapeutic target, however, RNA targets are generally underexploited due to a lack of understanding of the small molecules that bind RNA and the RNA motifs that bind small molecules. Herein, we describe the identification of the RNA internal loops derived from a 4096-member 3×3 nucleotide loop library that are the most specific and highest affinity binders to a series of four designer, drug-like benzimidazoles. These studies establish a potentially general protocol to define the highest affinity and most specific RNA motif targets for heterocyclic small molecules. Such information could be used to target functionally important RNAs in genomic sequence.

Hitting the target: A combined computational and experimental approach defines optimal and suboptimal RNA motifs as targets for small molecules through two‐dimensional combinatorial screening. The method, termed structure–activity relationships through sequencing (StARTS), uses information from the sequences of the RNA motifs selected to bind a ligand.

Herein, we report the identification of RNA hairpin loops that bind derivatives of kanamycin A, tobramycin, neamine, and neomycin B via two-dimensional combinatorial screening, a method that screens chemical and RNA spaces simultaneously. An arrayed aminoglycoside library was probed for binding to a 6-nucleotide RNA hairpin loop library (4096 members). Members of the loop library that bound each aminoglycoside were excised from the array, amplified and sequenced. Sequences were analyzed with our newly developed RNA Privileged Space Predictor (RNA-PSP) program, which analyzes selected sequences to identify statistically significant trends. RNA-PSP identified the following unique trends: 5′UNNNC3′ loops for the kanamycin A derivative (where N is any nucleotide); 5′UNNC3′ loops for the tobramycin derivative; 5′UNC3′ loops for the neamine derivative; and 5′UNNG3′ loops for the neomycin B derivative. The affinities and selectivities of a subset of the ligand–hairpin loop interactions were determined. The selected interactions have Kd values ranging from 10 nM to 605 nM. Selectivities ranged from 0.4 to >200-fold. Interestingly, the results from RNA-PSP are able to qualitatively predict specificity based on overlap between the RNA sequences selected for the ligands. These studies expand the information available on small molecule–RNA motif interactions, which could be useful to design ligands targeting RNA.

Increasing evidence supports a role for PKC␣ in growth arrest and tumor suppression in the intestinal epithelium. In contrast, the Id1 transcriptional repressor has pro-proliferative and tumorigenic properties in this tissue. Here, we identify Id1 as a novel target of PKC␣ signaling. Using a highly specific antibody and a combined morphological/biochemical approach, we establish that Id1 is a nuclear protein restricted to proliferating intestinal crypt cells. A relationship between PKC␣ and Id1 was supported by the demonstration that (a) down-regulation of Id1 at the crypt/villus junction coincides with PKC␣ activation, and (b) loss of PKC␣ in intestinal tumors is associated with increased levels of nuclear Id1. Manipulation of PKC␣ activity in IEC-18 nontransformed intestinal crypt cells determined that PKC␣ suppresses Id1 mRNA and protein via an Erk-dependent mechanism. PKC␣, but not PKC␦, also inhibited Id1 expression in colon cancer cells. Id1 was found to regulate cyclin D1 levels in IEC-18 and colon cancer cells, pointing to a role for Id1 suppression in the antiproliferative/tumor suppressive activities of PKC␣. Notably, Id1 expression was elevated in the intestinal epithelium of PKC␣-knock-out mice, confirming that PKC␣ regulates Id1 in vivo. A wider role for PKC␣ in control of inhibitor of DNA binding factors is supported by its ability to downregulate Id2 and Id3 in IEC-18 cells, although their suppression is more modest than that of Id1. This study provides the first demonstrated link between a specific PKC isozyme and inhibitor of DNA binding factors, and it points to a role for a PKC␣ 3 Erk ٜ Id1 3 cyclin D1 signaling axis in the maintenance of intestinal homeostasis.The intestinal epithelium is a continually self-renewing tissue, organized into well defined proliferative and functional compartments (1). Maintenance of tissue integrity relies on tight control of the balance between proliferative activity, differentiation, and apoptosis. Both positive and negative growth regulatory signaling pathways have been implicated in orchestrating the renewal process in this tissue, and disruption of these pathways and/or their downstream targets results in various diseases, including cancer. Increasing evidence points to the PKC enzyme system as a key regulator of intestinal homeostasis (2-6). PKC is a family of phospholipid-dependent serine-threonine kinases, consisting of at least 10 isozymes that act as central players in signal transduction. Members of the family can have tumor promoting (e.g. PKC␤II, -⑀, and -) or tumor suppressive (e.g. PKC␣ and -␦) activity, dependent on the isozyme and tissue context (2,5,7,8). Although several PKC isozymes have been implicated in the regulation of intestinal homeostasis (e.g. PKC␣, -␤II, -␦, -⑀, and -), increasing evidence points to PKC␣ as a key negative regulator of proliferation and tumorigenesis in this tissue (4, 9, 10).PKC␣ is activated precisely at the point of growth arrest in the crypts of both the small intestine and colon (11, 12). Consistent with a role i...