Technology : Overview

DNA-Scrunching During Transcription

The technology, based on Alternating Laser EXcitation (ALEX) single molecule fluorescence spectroscopy, is a unique SMD technology for ultrasensitive, specific, and highly multiplexed detection and quantification of target molecules such as proteins, RNAs (including microRNAs), and DNAs in complex samples. The technology also provides information about molecular interactions and dynamics to investigate biological mechanisms and analyze conformations of biomolecules using single molecule fluorescence resonance energy transfer (smFRET).

Target recognition molecules are tagged with different color fluorescent dyes. Coincident confocal detection of color-coded emission signals constitutes a positive target detection event, allowing molecular identification of freely diffusing molecules in solution and implementation of barcoding to detect numerous targets simultaneously.

The ALEX technology represents a particular breakthrough, allowing simultaneous stoichiometry- and smFRET-based analysis of molecular complexes and supramolecular assemblies. This can provide detailed views of molecular machines at work, e.g. as demonstrated for RNA polymerase performing “DNA-scrunching” during initial bacterial transcription^1,2, and allows multiplexed, barcoded detection of target molecules, highly useful for clinical diagnostics applications³.

How it works:
Two-Color ALEX
In its original, 2-color (2c) ALEX implementation, two high-affinity recognition molecules (e.g. antibodies, DNA oligomers), which are labeled with different color fluorescent dyes, bind to two mutually exclusive areas on the target molecule of interest. By counting the number of two different color coincident fluorescent bursts while the target diffuses through a femtoliter size confocal detection volume, the actual number (or concentration) of the target can be derived in a real time fashion after ‘virtual molecule sorting’. The dual-color coincidence detection by high speed laser alternation allows virtual exclusion of the majority of background noises, eliminating the need for washing steps, which, together with elimination of signal amplification steps, enables substantially improved accuracy of quantification with minimal sample and reagent requirements. Different distances between fluorescent donor (D) and acceptor (A) dyes allow differentiation between targets due to single molecule fluorescence resonance energy transfer (smFRET) which increases multiplexing power.

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Three and Four-Color ALEX
NTI recently successfully expanded the methodology from three (3c-ALEX) to four-colors (4c-ALEX)³. Molecule sorting in multi-dimensional space substantially extends ALEX single-well multiplexing capabilities and permits differentiation of numerous targets simultaneously. Exploiting smFRET to monitor distances between fluorescent donors and acceptors incorporated at specific sites on individual molecules allows implementation of fluorophore “barcoding” (NTI U.S. Provisional Patent Application No. 61/576,897). 4c-ALEX increases the number of different types of molecules to be detected in a sample within a single well theoretically up to 100 (25 have been demonstrated³). Furthermore, NTI achieved ultra-sensitive, amplification-free detection of micro-RNA at clinically-relevant concentrations (NTI U.S. Provisional Patent Application No. 61/552,404), and outlined an approach for ultra-sensitive detection of low abundance proteins (NTI U.S. Provisional Patent Application No. 61/576,889).

1. A. N. Kapanidis et al., Mol Cell 20, 347 (2005)
2. A. N. Kapanidis et al., Science 314, 1144 (2006)
3. S. W. Yim et al., Clin Chem 58, 707 (2012)