Comparative Study of Thermal Stability and On/Off Fluorescent Signaling Characteristics of Self-Quenching Smart Probes
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RESEARCH ARTICLE-CHEMISTRY
Comparative Study of Thermal Stability and On/Off Fluorescent Signaling Characteristics of Self-Quenching Smart Probes Sulayman A. Oladepo1
· Basiru O. Yusuf1 · Hussain Alzaindeen1
Received: 9 November 2019 / Accepted: 13 August 2020 © King Fahd University of Petroleum & Minerals 2020
Abstract The fluorescence signal levels produced by smart probes (SPs) largely depend on the type of fluorophore label used and the quenching efficiency of the quencher. However, there has been little emphasis on the effects of terminal location of the fluorophore and the modifications used to attach the fluorophore to the oligonucleotide chain. To this end, we have conducted a comparative study of the fluorescent signaling characteristics of six SPs consisting of three fluorophores: 6-FAM, ATTO488 and TAMRA, each of which was attached to either the 3 end or the 5 end of the oligonucleotide probe to make two SPs each. In each case, the fluorophore was paired with guanosine quenchers and we determined the fluorescence signaling characteristic of each SP by hybridizing it with the complementary target and measuring the fluorescence signal produced. Our results showed that the melting temperatures (T m ’s) of each two SPs consisting of the same fluorophore were different before hybridization with targets but were similar once hybridized. We also found that each two SPs made of the same fluorophore gave different fluorescence signal levels. Our data also revealed a subtle correlation between T m differences and fluorescence intensity differences for each pair of SPs consisting of the same fluorophore. We interpret the observed differences in T m ’s and fluorescence signals in terms of possible structural differences in the linker moieties used for coupling the fluorophores to the 3 and 5 ends as well as possible differences in the efficiency of coupling the fluorophore during the probe synthesis. Keywords Smart probe · Fluorescent hairpin probes · Melting temperature · Thermal stability · Fluorescence signaling characteristics
1 Introduction Smart probes (SPs) constitute a class of fluorescent hairpin probes with a fluorophore on one end and guanosine quenchers on the other end of the oligonucleotide chain [1–5]. Their constrained hairpin structure consists of a loop that is perfectly complementary to the target sequence of interest, while the stem consists of two complementary strands terminated by a fluorophore on one end and neighboring guanosine quencher on the other [1, 6]. Guanosine is used as a quencher because of its low oxidation potential [4, 7, 8]. The Electronic supplementary material The online version of this article (https://doi.org/10.1007/s13369-020-04860-5) contains supplementary material, which is available to authorized users.
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Sulayman A. Oladepo [email protected] Department of Chemistry, King Fahd University of Petroleum and Minerals, Dhahran 31261, Kingdom of Saudi Arabia
close proximity of guanosine and the fluorophore promotes fluorescence quenching via photoinduced i
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