Quenching of Fluorophore-Labeled DNA Oligonucleotides by Divalent Metal Ions:  Implications for Selection, Design, and Applications of Signaling Aptamers and Signaling Deoxyribozymes

Abstract

Recent years have seen a dramatic increase in the use of fluorescence-signaling DNA aptamers and deoxyribozymes as novel biosensing moieties. Many of these functional single-stranded DNA molecules are either engineered to function in the presence of divalent metal ion cofactors or designed as sensors for specific divalent metal ions. However, many divalent metal ions are potent fluorescence quenchers. In this study, we first set out to examine the factors that contribute to quenching of DNA-bound fluorophores by commonly used divalent metal ions, with the goal of establishing general principles that can guide future exploitation of fluorescence-signaling DNA aptamers and deoxyribozymes as biosensing probes. We then extended these studies to examine the effect of specific metals on the signaling performance of both a structure-switching signaling DNA aptamer and an RNA-cleaving and fluorescence-signaling deoxyribozyme. These studies showed extensive quenching was obtained when using divalent transition metal ions owing to direct DNA-metal ion interactions, leading to combined static and dynamic quenching. The extent of quenching was dependent on the type of metal ion and the concentration of supporting monovalent cations in the buffer, with quenching increasing with the number of unpaired electrons in the metal ion and decreasing with the concentration of monovalent ions. The extent of quenching was independent of the fluorophore, indicating that quenching cannot be alleviated simply by changing the nature of the fluorescent probe. Our results also show that the DNA sequence and the local secondary structure in the region of the fluorescent tag can dramatically influence the degree of quenching by divalent transition metal ions. In particular, the extent of quenching is predominantly determined by the fluorophore location with respect to guanine-rich and duplex regions within the strand sequence. Examination of the effect of both the type and concentration of metal ions on the performance of a fluorescence-signaling aptamer and a signaling deoxyribozyme confirms that judicious choice of divalent transition metal ions is important in maximizing signals obtained from such systems.

Altering the Divalent Metal Ion Preference of RNase E

“…Titration experiments using either 5=-32 P-BR10 or 5=P-BR14FD showed that the optimal ionic conditions for maximal activity of the WT enzyme were 10 mM MgCl 2 , similar to the value reported by Redko et al (6), or 400 M MnSO 4 (data not shown). Quenching of fluorescence by Mn 2ϩ or other transition metal ions is a well-known problem (26). To circumvent this in assays using 5=-P-BR14FD, 80 mM KCl was added to partially mask the backbone charge on the substrate, and divalent ion concentrations were set at 10 mM Mg 2ϩ Ϯ 0.1 mM Mn 2ϩ (i.e., a 100:1 ratio) despite the latter being suboptimal for Mn 2ϩ .…”

Detection of Bacteria Using Fluorogenic DNAzymes

“…FS1 was obtained from Keck Oligo Synthesis Facilities at Yale University, deprotected and purified by gel electrophoresis following a previously established protocol. [17][18][19][20][21][22][23][24] EC1, SS1 and LT1 were purchased from Integrated DNA Technologies and purified by gel electrophoresis. vortexing and place the tubes in -20 °C freezer for 30 min.…”

“…1) and investigated the use of RFDs as analytical tools. [17][18][19][20][21][22][23][24][25][26][27][28][29] RFDs catalyze the cleavage of a DNA-RNA chimeric substrate at a single ribonucleotide junction (R) that is flanked by a fluorophore (F) and a quencher (Q). The close proximity of F and Q renders the uncleaved substrate minimal fluorescence.…”

Aptasensors for biosecurity applications

“…[48,49] Briefly, a 3-piece DNA sandwich was first formed to bring a fluorophore-labeled DNA oligonucleotide (F-DNA) in close proximity to a quencher oligonucleotide (Q-DNA). In the presence of the target molecule, the unmodified oligonucleotide (MAP), which contains the target binding aptamer sequences, will fold to enable tight binding to its target (Fig.…”