Ultrasensitive detection of DNA and protein markers in cancer cells.

Cancer cells differ from normal cells in various parameters, and these differences are caused by genomic mutations and consequential altered gene expression. The genetic and functional heterogeneity of tumor cells is a major challenge in cancer research, detection, and effective treatment. As such, the use of diagnostic methods is important to reveal this heterogeneity at the single-cell level.

Analysis of RNA localization and metabolism in single live bacterial cells: achievements and challenges.

Studies of gene expression at the single cell level in live bacterial cells represent a new and fertile area of research providing real-time, spatially specified information that cannot be obtained by techniques relying on large cell populations. Before recently, most single-cell studies have been concerned with gene expression at the protein level and explored the spatiotemporal localization and dynamics of different bacterial proteins. However, to fully understand the complex process of gene expression, it is necessary to visualize and quantify RNA molecules in the cellular environment.

Visualization of induced RNA in single bacterial cells.

Visualization of RNA in live cells is a challenging task due to the transient character of most RNA molecules and the lack of adequate methods to label RNA noninvasively. Here, we describe a system for regulated RNA synthesis and visualization of RNA in live Escherichia coli cells based on protein complementation. This method allows for labeling RNA with a relatively small protein complex that becomes fluorescent only when bound to an RNA.

Spatiotemporal patterns and transcription kinetics of induced RNA in single bacterial cells.

Bacteria have a complex internal organization with specific localization of many proteins and DNA, which dynamically move during the cell cycle and in response to changing environmental stimuli. Much less is known, however, about the localization and movements of RNA molecules. By modifying our previous RNA labeling system, we monitor the expression and localization of a model RNA transcript in live Escherichia coli cells.

Ab initio RNA folding by discrete molecular dynamics: from structure prediction to folding mechanisms.

RNA molecules with novel functions have revived interest in the accurate prediction of RNA three-dimensional (3D) structure and folding dynamics. However, existing methods are inefficient in automated 3D structure prediction. Here, we report a robust computational approach for rapid folding of RNA molecules.

Visualization of RNA using fluorescence complementation triggered by aptamer-protein interactions (RFAP) in live bacterial cells.

This unit describes a method allowing RNA visualization in live cells. The method is based on fluorescent protein complementation regulated by RNA-aptamer/RNA-binding protein interactions. Based on these two principles, a fluorescent ribonucleoprotein complex is assembled inside the cell only in response to the presence of the aptamer sequence on the target RNA.

Profluorescent protein fragments for fast bimolecular fluorescence complementation in vitro.

Here, we present a protocol for isolating the large N-terminal fragment of enhanced green fluorescent protein (EGFP) with a preformed chromophore. By itself, the chromophore-containing EGFP fragment exhibits very weak fluorescence, but it rapidly becomes brightly fluorescent upon complementation with the corresponding small, C-terminal EGFP fragment. Each EGFP fragment is cloned and overexpressed in E.

RNA visualization in live bacterial cells using fluorescent protein complementation.

We describe a technique for the detection and localization of RNA transcripts in living cells. The method is based on fluorescent-protein complementation regulated by the interaction of a split RNA-binding protein with its corresponding RNA aptamer. In our design, the RNA-binding protein is the eukaryotic initiation factor 4A (eIF4A).

Fast complementation of split fluorescent protein triggered by DNA hybridization.

Fluorescent proteins have proven to be excellent reporters and biochemical sensors with a wide range of applications. In a split form, they are not fluorescent, but their fluorescence can be restored by supplementary protein-protein or protein-nucleic acid interactions that reassemble the split polypeptides. However, in prior studies, it took hours to restore the fluorescence of a split fluorescent protein because the formation of the protein chromophore slowly occurred de novo concurrently with reassembly.

GC/AT-content spikes as genomic punctuation marks.

Large-scale analysis of the GC-content distribution at the gene level reveals both common features and basic differences in genomes of different groups of species. Sharp changes in GC content are detected at the transcription boundaries for all species analyzed, including human, mouse, rat, chicken, fruit fly, and worm. However, two substantially distinct groups of GC-content profiles can be recognized: warm-blooded vertebrates including human, mouse, rat, and chicken, and invertebrates including fruit fly and worm.

Differential display in the time of microarrays.

A brief overview of major methods used for genome-wide expression profiling is presented. Special attention is devoted to ordered differential display, subtractive hybridization and DNA microarrays. Future prospects of comparative gene expression studies using combinations of differential display methods and microarray technology are outlined.

Stem-loop oligonucleotides: a robust tool for molecular biology and biotechnology.

The specific structural features of stem-loop (hairpin) DNA constructs provide increased specificity of target recognition. Recently, several robust assays have been developed that exploit the potential of structurally constrained oligonucleotides to hybridize with their cognate targets. Here, I review new diagnostic approaches based on the formation of stem-loop DNA oligonucleotides: molecular beacon methodology, suppression PCR approaches and the use of hairpin probes in DNA microarrays.