Sequence-specific Fluorescence Research Articles

Sequence-Specific Fluorescence Turn-On Sensing of RNA by DNA Probes Incorporating the Tricyclic Cytidine Analogue DEAtC.

Sequence-specific fluorescent probes for RNA are widely used in microscopy applications such as fluorescence in situ hybridization and a growing number of newer approaches to live-cell RNA imaging. The sequence specificity of most of these approaches relies on differential hybridization of the probe to the correct target. Competing sequences with only one or two base mismatches are prone to causing off-target recognition. Here, we report the sequence-specific fluorescent detection of model RNA targets using a tricyclic cytidine analogue DEAtC that is included as a surrogate for natural cytidine in DNA probe strands and that reports directly on Watson-Crick base pairing. The DEAtC-containing DNA oligonucleotide probes exhibit an average 8-fold increase in fluorescence intensity when hybridized to matched RNA with DEAtC base paired with G and little fluorescence turn-on when DEAtC is base paired with A. Duplex structure determination by NMR, time-resolved fluorescence studies, and Stern-Volmer quenching experiments suggest that the combination of greater π stacking and narrower grooves in the A-form DNA-RNA heteroduplex provides additional shielding and favorable electronic interactions between bases, explaining why DEAtC's fluorescence turn-on response to RNA targets is typically 3-fold greater than for DNA targets.

Isothermal amplification using sequence-specific fluorescence detection of SARS coronavirus 2 and variants in nasal swabs.

Coronavirus disease 2019 is a public health challenge requiring rapid testing for the detection of infections and transmission. Nucleic acid amplification tests targeting SARS coronavirus 2 (CoV2) are used to detect CoV2 in clinical samples. Real-time reverse transcription quantitative PCR is the standard nucleic acid amplification test for CoV2, although reverse transcription loop-mediated isothermal amplification is used in diagnostics. The authors demonstrate a sequence-specific reverse transcription loop-mediated isothermal amplification-based nucleic acid amplification assay that is finished within 30 min using minimally processed clinical nasal swab samples and describe a fluorescence-quenched reverse transcription loop-mediated isothermal amplification assay using labeled primers and a quencher oligonucleotide. This assay can achieve rapid (30 min) and sensitive (1000 plaque-forming units/ml) fluorescence detection of CoV2 (WA1/2020), B.1.1.7 (Alpha) and variants of concern Delta (B.1.617.2) and Omicron (B.1.1.529) in nasal samples.

Combining dense and sparse labeling in optical DNA mapping.

Optical DNA mapping (ODM) is based on fluorescent labeling, stretching and imaging of single DNA molecules to obtain sequence-specific fluorescence profiles, DNA barcodes. These barcodes can be mapped to theoretical counterparts obtained from DNA reference sequences, which in turn allow for DNA identification in complex samples and for detecting structural changes in individual DNA molecules. There are several types of DNA labeling schemes for ODM and for each labeling type one or several types of match scoring methods are used. By combining the information from multiple labeling schemes one can potentially improve mapping confidence; however, combining match scores from different labeling assays has not been implemented yet. In this study, we introduce two theoretical methods for dealing with analysis of DNA molecules with multiple label types. In our first method, we convert the alignment scores, given as output from the different assays, into p-values using carefully crafted null models. We then combine the p-values for different label types using standard methods to obtain a combined match score and an associated combined p-value. In the second method, we use a block bootstrap approach to check for the uniqueness of a match to a database for all barcodes matching with a combined p-value below a predefined threshold. For obtaining experimental dual-labeled DNA barcodes, we introduce a novel assay where we cut plasmid DNA molecules from bacteria with restriction enzymes and the cut sites serve as sequence-specific markers, which together with barcodes obtained using the established competitive binding labeling method, form a dual-labeled barcode. All experimental data in this study originates from this assay, but we point out that our theoretical framework can be used to combine data from all kinds of available optical DNA mapping assays. We test our multiple labeling frameworks on barcodes from two different plasmids and synthetically generated barcodes (combined competitive-binding- and nick-labeling). It is demonstrated that by simultaneously using the information from all label types, we can substantially increase the significance when we match experimental barcodes to a database consisting of theoretical barcodes for all sequenced plasmids.

DNA-Templated Fluorescent Silver Nanoclusters Inhibit Bacterial Growth While Being Non-Toxic to Mammalian Cells.

Silver has a long history of antibacterial effectiveness. The combination of atomically precise metal nanoclusters with the field of nucleic acid nanotechnology has given rise to DNA-templated silver nanoclusters (DNA-AgNCs) which can be engineered with reproducible and unique fluorescent properties and antibacterial activity. Furthermore, cytosine-rich single-stranded DNA oligonucleotides designed to fold into hairpin structures improve the stability of AgNCs and additionally modulate their antibacterial properties and the quality of observed fluorescent signals. In this work, we characterize the sequence-specific fluorescence and composition of four representative DNA-AgNCs, compare their corresponding antibacterial effectiveness at different pH, and assess cytotoxicity to several mammalian cell lines.

DNA/RNA Fluorescence Imaging by Synthetic Nucleic Acids.

Fluorescence imaging of nucleic acids is a key technology needed to understand gene expression and the resulting changes in cellular function. This chapter focuses on sequence-specific fluorescence imaging of nucleic acids in cells using fluorescent nucleic acids. The design and preparation of fluorescent nucleic acids and their application to fluorescence imaging of intracellular nucleic acids are introduced.

Sequence-Specific Post-Synthetic Oligonucleotide Labeling for Single-Molecule Fluorescence Applications.

The sequence-specific fluorescence labeling of nucleic acids is a prerequisite for various methods including single-molecule Förster resonance energy transfer (smFRET) for the detailed study of nucleic acid folding and function. Such nucleic acid derivatives are commonly obtained by solid-phase methods; however, yields decrease rapidly with increasing length and restrict the practicability of this approach for long strands. Here, we report a new labeling strategy for the postsynthetic incorporation of a bioorthogonal group into single stranded regions of both DNA and RNA of unrestricted length. A 12-alkyne-etheno-adenine modification is sequence-selectively formed using DNA-templated synthesis, followed by conjugation of the fluorophore Cy3 via a copper-catalyzed azide-alkyne cycloaddition (CuAAC). Evaluation of the labeled strands in smFRET measurements shows that the strategy developed here has the potential to be used for the study of long functional nucleic acids by (single-molecule) fluorescence or other methods. To prove the universal use of the method, its application was successfully extended to the labeling of a short RNA single strand. As a proof-of-concept, also the labeling of a large RNA molecule in form of a 633 nucleotide long construct derived from the Saccharomyces cerevisiae group II intron Sc.ai5γ was performed, and covalent attachment of the Cy3 fluorophore was shown with gel electrophoresis.

MicroRNA-Responsive Drug Release System for Selective Fluorescence Imaging and Photodynamic Therapy In Vivo.

Photodynamic therapy (PDT) is a noninvasive strategy to treat diseases by light-triggered activation of a photosensitizer (PS). One aim of the recent researches on PDT is to overcome the limitation of conventional PDT by improving selective activation of PS on targeted region. Here, a microRNA (miRNA)-responsive drug activation system is developed which focuses on the role of endogenous miRNA as an internal cancer specific stimulus for initiating drug release in cancer treatment. The present system consists of PS chlorin e6 (Ce6) conjugated to peptide nucleic acid (PNA) having complementary sequence to cancer specific miRNA and dextran coated reduced graphene oxide nanocolloid (Dex-RGON). In the presence of oncogenic miR-21 in cancer cells, Ce6-PNA drug gets hybridized with miR-21, resulting in the release of Ce6-PNA from Dex-RGON and subsequent recovery of Ce6 fluorescence and activation of Ce6 as a photosensitizer under near IR irradiation. It is demonstrated that the Ce6-PNA/Dex-RGON complex shows sequence-specific fluorescence in response to miR-21 and selective cytotoxic effect for tumor growth inhibition. The present study will pave a new way for utilizing PDT in cancer treatment with tightly regulated activation of a photosensitizer by oncogenic miRNA as an internal stimulus to reduce potential risk associated with conventional PDT.

Abstract A26: Telomere length in normal and tumor breast tissue from BRCA2 mutation carriers and sporadic breast cancer cases

Abstract Germline mutations in the BRCA genes are associated with highly increased risk of breast and ovarian cancers and to a lesser extent to prostate and pancreatic cancers. Great number of mutations with variable impact on cancer risk and progression are known in these genes worldwide. A single founder mutation has been detected in each of the BRCA genes in the Icelandic population, making it feasible to study the influence of a single mutation at a population level. The BRCA2999del5 mutation is more frequent and can be found in approximately 6-7% of female breast cancer patients and 40% of male breast cancer patients in Iceland. The mutation leads to a non-functional protein product and is associated with complex chromosomal changes in tumor tissue. Furthermore, the BRCA2999del5 mutation has been associated with poor prognosis, both in breast and prostate cancers, although mutation carriers seem to differ with respect to age of onset and severity of disease. Dysfunctional telomere maintenance can lead to excessive telomere shortening which causes chromosome instability. It is therefore a driving force behind cancer progression and is considered a hallmark of many human cancers. Telomere shortening has been shown to be an early and a common molecular alteration in epithelial cancers, including breast cancers. Previous results from our laboratory, amongst others, have shown that BRCA2 is associated with telomere protection and maintenance. Telomere dysfunction induced foci (TIFs) formation was detected in cell lines derived from heterozygous BRCA2999del5 mutation carriers1. Telomere length has been proposed as a possible predictive factor for cancer risk for various cancers. In the case of breast cancer and BRCA mutation carriers, the results have been conflicting. The aim of the study was to investigate if telomere length in normal and tumor breast tissue is correlated with breast cancer progression and survival in a well-defined group of BRCA2 mutation carriers and sporadic breast cancer cases. The study group consisted of breast cancer patients carrying the BRCA2999del5 Icelandic founder mutation and sporadic cases, matched with respect to age and year of diagnosis. Tumor and normal adjacent tissue samples from these patients were embedded in paraffin and Quantitative Fluorescence in Situ Hybridization method (Q-FISH) was used to carry out telomere length measurements. Telomeres were visualized using a telomeric sequence-specific fluorescence peptide nucleic acid (PNA) probe. A centromere-specific PNA probe was used as an inner control for hybridization efficiency. The results show a clear difference in telomere length of different cell types in normal breast tissue with inner epithelial cells having shorter telomeres than both myoepithelial cells and fibroblasts. These findings support previously reported data in the literature2. The majority of examined tumors have very short telomeres. In mutation carriers, there are indications that less variable telomere length in normal adjacent tissue is associated with reduced breast cancer specific survival. This seems to be more pronounced in the mutation carrier group compared to the sporadic group. Telomere length in adjacent normal tissue appears to be shorter in cases with luminal type than with basal-like tumors.

Sequence-specific recognition of DNA minor groove by an NIR-fluorescence switch-on probe and its potential applications.

In molecular biology, understanding the functional and structural aspects of DNA requires sequence-specific DNA binding probes. Especially, sequence-specific fluorescence probes offer the advantage of real-time monitoring of the conformational and structural reorganization of DNA in living cells. Herein, we designed a new class of D2A (one-donor-two-acceptor) near-infrared (NIR) fluorescence switch-on probe named quinone cyanine–dithiazole (QCy–DT) based on the distinctive internal charge transfer (ICT) process for minor groove recognition of AT-rich DNA. Interestingly, QCy–DT exhibited strong NIR-fluorescence enhancement in the presence of AT-rich DNA compared to GC-rich and single-stranded DNAs. We show sequence-specific minor groove recognition of QCy–DT for DNA containing 5′-AATT-3′ sequence over other variable (A/T)4 sequences and local nucleobase variation study around the 5′-X(AATT)Y-3′ recognition sequence revealed that X = A and Y = T are the most preferable nucleobases. The live cell imaging studies confirmed mammalian cell permeability, low-toxicity and selective staining capacity of nuclear DNA without requiring RNase treatment. Further, Plasmodium falciparum with an AT-rich genome showed specific uptake with a reasonably low IC50 value (<4 µM). The ease of synthesis, large Stokes shift, sequence-specific DNA minor groove recognition with switch-on NIR-fluorescence, photostability and parasite staining with low IC50 make QCy–DT a potential and commercially viable DNA probe.

Rapid Detection of TEM-Type Extended-Spectrum β-Lactamase (ESBL) Mutations Using Lights-On/Lights-Off Probes with Single-Stranded DNA Amplification

Rapid identification of specific TEM-type β-lactamase genes in bacterial infections is important for determining appropriate clinical treatment. We report here the design and initial testing of a molecular diagnostic assay capable of amplifying a large segment of the blaTEM gene, as well as detecting widely spaced extended-spectrum β-lactamase (ESBL) mutations and inhibitor-resistant TEM (IRT) mutations (eg, clavulanic acid resistance). Single-stranded DNA is generated using linear-after-the-exponential PCR (LATE-PCR) and is analyzed at the endpoint, using a set of four fluorescently labeled and four quencher-labeled probes in a single closed tube. These lights-on/lights-off probes work in concert to generate sequence-specific fluorescence contours over a temperature range from 25°C to 75°C. Mutant sequences from synthetic TEM gene variants and from TEM gene variants in bacterial strains generated large increases in fluorescent signal relative to that from the reference sequence for TEM-1. Clinical use of this convenient, single-closed-tube assay would make it possible to rapidly distinguish ESBL from non-ESBL variants and thereby to begin early treatment with suitable antibiotics.

Sequence-Specific Fluorescence Detection of DNA by Polyamide−Thiazole Orange Conjugates

Fluorescent methods to detect specific double-stranded DNA sequences without the need for denaturation may be useful in the field of genetics. Three hairpin pyrrole-imidazole polyamides 2-4 that target their respective sequences 5'-WGGGWW-3', 5'-WGGCCW-3', and 5'-WGWWCW-3' (W = A or T) were conjugated to thiazole orange dye at the C-termini to examine their fluorescence properties in the presence and absence of match duplex DNA. The conjugates fluoresce weakly in the absence of DNA but showed significant enhancement (>1000-fold) upon the addition of 1 equiv of match DNA and only slight enhancement with the addition of mismatch DNA. The polyamide-dye conjugates bound specific DNA sequences with high affinity (Ka > 10(8) M(-1)) and unwound the DNA duplex through intercalation (unwinding angle, phi, approximately 8 degrees). This new class of polyamides provides a method to specifically detect DNA sequences without denaturation.

Aptamers switch on fluorescence of triphenylmethane dyes.

MG and SRB aptamers, which are short RNA sequences originally selected only for binding to malachite green or sulforhodamine B, can greatly enhance the fluorescence of normally nonfluorescent triphenylmethane dyes. MG aptamer enhances the quantum yields of malachite green (MG) and a novel rigidized derivative, indolinyl malachite green (IMG) by >2000-fold. SRB aptamer brightens patent blue V and VF by >90-fold. These enhancements are specific because MG aptamer has no effect on patent blue dyes and SRB aptamer has little or no effect on MG and IMG. Such sequence-specific fluorescence labeling of short RNA motifs is a first step toward genetically encodable fusion tags for imaging selected RNAs in vitro and in cells.

A novel method for SNP detection using a new duplex-specific nuclease from crab hepatopancreas.

We have characterized a novel nuclease from the Kamchatka crab, designated duplex-specific nuclease (DSN). DSN displays a strong preference for cleaving double-stranded DNA and DNA in DNA-RNA hybrid duplexes, compared to single-stranded DNA. Moreover, the cleavage rate of short, perfectly matched DNA duplexes by this enzyme is essentially higher than that for nonperfectly matched duplexes of the same length. Thus, DSN differentiates between one-nucleotide variations in DNA. We developed a novel assay for single nucleotide polymorphism (SNP) detection based on this unique property, termed "duplex-specific nuclease preference" (DSNP). In this innovative assay, the DNA region containing the SNP site is amplified and the PCR product mixed with signal probes (FRET-labeled short sequence-specific oligonucleotides) and DSN. During incubation, only perfectly matched duplexes between the DNA template and signal probe are cleaved by DSN to generate sequence-specific fluorescence. The use of FRET-labeled signal probes coupled with the specificity of DSN presents a simple and efficient method for detecting SNPs. We have employed the DSNP assay for the typing of SNPs in methyltetrahydrofolate reductase, prothrombin and p53 genes on homozygous and heterozygous genomic DNA.

Different Real-Time PCR Formats Compared for the Quantitative Detection of Human Cytomegalovirus DNA

The aim of this study was to compare the ABI PRISM 7700 Sequence Detection System and the LightCycler to develop a quantitative real-time PCR assay for the detection of human cytomegalovirus (HCMV) DNA suitable for routine hospital application. We used one exonuclease probe and five different hybridization probe sets as sequence-specific fluorescence detection formats. For the exonuclease assay and two hybridization probe sets, reproducibility and the detection limit were determined. To keep the total assay time to a minimum, we gradually shortened individual reaction steps on both instruments. The exonuclease assay can be interchangeably performed on the 7700 and the LightCycler. No change of reaction conditions is required, except for the addition of bovine serum albumin to the LightCycler reaction. The shortest possible total assay time is 80 min for the ABI PRISM 7700 Sequence Detection System and 20 min for the LightCycler. When the LightCycler is used, the exonuclease probe can be replaced by a set of hybridization probes. All assays presented here detected HCMV DNA in a linear range from 10(1) to 10(7) HCMV genome equivalents/assay (r >0.995) with low intraassay (<5%) and interassay (<10%) variation. The ABI PRISM 7700 Sequence Detection System as well as the LightCycler are useful instruments for rapid and precise online PCR detection. Moreover, the two principles of fluorescence signal production allow HCMV quantification with the same accuracy.