Quenched probes for highly specific detection of cellular RNAs

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Nucleic acid-based RNA detection is a promising field in molecular biotechnology that is leading to the rapid and accurate identification of microorganisms, diagnosis of infections and imaging of gene expression. The specificity of short synthetic DNA probes raises the hope of distinguishing small differences in sequence, ultimately achieving single nucleotide resolution. Recent work using quenched fluorescently labeled oligonucleotide probes as sensors for RNA in bacterial and human cells has overcome several difficult hurdles on the way to these goals, including delivery of probes to live cells, accessing RNA sites containing a high degree of secondary structure, and eliminating many sources of background. Two new classes of quenched oligonucleotide probes, molecular beacons and quenched auto-ligation probes, have shown the most promise for in situ RNA detection. High-specificity detection, at the single-nucleotide resolution level, is now possible in solution with these classes of probes. However, for applications in intact cells, signal and background issues still need to be addressed before the full potential of these methods is achieved.

Introduction

Methods for distinguishing RNA sequences in cells with single nucleotide specificity are of great interest because of their myriad applications in the fields of biochemistry, molecular biology, bioinformatics and pathology. Ribosomal RNA (rRNA) has long been a target for in situ studies because of its abundance and its accessibility to oligonucleotide probes. More recently, there has also been substantial research on detecting messenger RNA (mRNA) in cells for obtaining diagnostic or gene expression data.

There is a long history of development of lengthy polymeric DNA or RNA probes for in situ hybridization to RNA, and the field continues to move rapidly. However, long probes made enzymatically (hundreds or thousands of nucleotides in length) are not able to distinguish the smallest differences in rRNA or mRNA sequence. Because of the importance of small sequence differences in distinguishing between strains of bacteria, detecting drug resistance and evaluating the early stages of cancer, there is increasing interest in the development of small oligonucleotide-based probes for in situ imaging. RNA sequences generally can be identified by using fluorescently labeled oligonucleotide probes (typically 15–30 nucleotides in length), which hybridize to a complementary sequence of interest. Such probes are directly applicable to genus-, or in some cases, species-specific identification of organisms in wastewater, food and water supplies, bioreactors and clinical diagnostics [1]. However, probing for single nucleotide polymorphisms (SNPs) is often difficult. Two or more mismatches are typically required for highly accurate detection using an in situ hybridization probe of 15–20 nucleotides; thus, identification of SNPs in bacterial or human cells is usually impossible with traditional methods [2].

However, the need for such specificity is clear. Antibiotic resistance in bacteria is often caused by single base mutations in the rRNA sequence [3]. Closely related bacteria, such as Escherichia coli and Salmonella species, often have >97% similarity in their rRNA sequences, with the few variations typically found as SNPs rather than variable domains [4]. Single nucleotide mutations in human cells, such as the well-characterized H-ras point mutation, can lead to activation of oncogenes [5]. Unfortunately, the most reliable strategy for highly specific RNA identification, reverse-transcriptase complimentary DNA (cDNA) synthesis, followed by polymerase chain reaction (PCR) amplification, and often sequencing, is labor intensive, time consuming and requires expensive reagents and equipment [6]. This approach is often not practical for applications that require high-throughput or rapid results, such as clinical diagnostics or genomic analyses, and so a great deal of recent research has focused on developing highly specific methods to probe RNA sequences in cells directly.

Section snippets

Standard fluorescent (non-quenched) oligonucleotide probes in fixed cells

The traditional probes for achieving selective RNA detection in situ are standard oligonucleotides carrying one or more fluorescent labels, and with no quenching groups. For example, the simplest and most widely used method for detecting rRNA in cells, fluorescence in situ hybridization (FISH), is typically performed with rRNA-targeted oligonucleotide probes, 15–30 bases long, end-labeled with a fluorophore. To remove unbound probes that emit nonspecific signals, careful washing must be

Quenched oligonucleotide probes

Over the past decade, many of the improvements in FISH technology have focused on developing ways to (i) reduce background signal and (ii) eliminate the need for washing steps so that live cells can be used. These goals have been achieved by using quenched oligonucleotide probes. Similar to normal FISH probes, quenched oligonucleotide probes are short DNA probes but they are modified with a fluorescence quencher (such as dabcyl) in addition to the fluorophore. Owing to structural or

Molecular beacons

Molecular beacons were first developed for monitoring nucleic-acid amplification assays [16] but their potential as in situ probes was quickly realized. A molecular beacon is an oligonucleotide that undergoes a conformational change upon hybridizing to a complementary target, resulting in a fluorescent signal. In its native state, the probe is a hairpin with the target sequence in the loop and a sequence that is non-complementary to the target in the stem. A fluorophore is attached to one end

Quenched auto-ligation (QUAL) probes

Similar to molecular beacons, QUAL probes were designed to be highly sequence specific for RNAs and show a simple on or off signal in the presence or absence of the sequence of interest. Unlike molecular beacons, the fluorescence signal of QUAL probes is the result of a chemical reaction, not a conformational change. QUAL probes consist of a probe pair: one probe contains an internal fluorophore and a 5′-terminal quencher (typically dabsyl) attached by an electrophilic sulfonic acid linkage;

Limitations of current methodologies

The most fundamental problems of cellular RNA detection methods that must be addressed are those of signal and background. Some improvements and modifications to existing strategies will be required to fully maximize signal (for detection of low-copy-number RNAs) and to minimize background (to avoid false positives).

Positive signal can be limited by the intensity of the fluorescent label on the unquenched probe, by the number of target molecules, and by the numbers of probes introduced into the

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