Tetracycline bivalent aptamer sequence and non-enzyme label-free detection method
A tetracycline aptamer and tetracycline technology, applied in the field of molecular biology technology, chemical analysis and detection, can solve the problems that detection is difficult to get rid of large-scale equipment, increase analysis cost, complex reaction system and other problems, achieve good practical value and facilitate promotion , Sensitive effect
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Embodiment 1
[0044] Embodiment 1 Experimental principle
[0045] The 8nt tetracycline aptamer clipped by Gu and Raston et al. is a G-rich sequence, and the sequence composition has the potential to form a split G-quadruplex. Since the G-quadruplex structure can strongly excite ThT fluorescence, this application is approved Rational design of the sequence, without changing its own base, introduces an 8nt single sequence into the repeat unit to form a new sequence based on a 16nt bivalent tetracycline aptamer, endowing it with better G-quadruplex formation ability. Furthermore, by introducing a base unit of appropriate length between the motif intervals of the bivalent aptamer, the sequence can be folded more easily to form a stable G-quadruplex structure, thereby strongly exciting ThT fluorescence in the absence of a target. After the target, the high affinity between tetracycline and the sequence destroys the G-quadruplex formation conditions, ThT fluorescence cannot be effectively excite...
Embodiment 2 4
[0046] Example 2 Tetracycline bivalent aptamer sequence design
[0047] The tetracycline bivalent aptamer sequences used in the experiment are shown in Table 1. The basis of its rational design is that the 8nt tetracycline aptamer is a G-rich sequence, which has the potential to form a cleavage G-quadruplex. Sequence (SEQ ID NO.1), which endows it with better G-quadruplex formation ability; secondly, base units of appropriate length are introduced between the motif intervals of the bivalent aptamer, so that the sequence can be folded more easily to form a stable G - Quadruplex structure (SEQ ID NO. 2-9).
[0048] Table 1 Tetracycline bivalent aptamer sequence design
[0049]
[0050]
[0051] Note: The underlined content is the inserted base sequence
Embodiment 3 4
[0052] Example 3 Evaluation of Tetracycline Bivalent Aptamer Sequence to Stimulate ThT Ability
[0053] Dissolve the sequences in Table 1 to 100 μM in ultrapure water, thermally melt at 95°C for 5 minutes in a PCR machine, and cool naturally to room temperature for later use. Add the sequence to 20 mM Tris-K pH=7.4 + buffer, to a final concentration of 1 μM, before detection, add ThT to the system at a final concentration of 1 μM, and mix well. Put 100 μL detection system in the fluorescence spectrophotometer immediately, select the fluorescence measurement mode, set the emission and excitation slit width to 10nm, the excitation wavelength of fluorescent molecules to 443nm, and collect the maximum emission light intensity at 490nm.
[0054] the result shows( figure 2 a) Compared with the tetracycline aptamer sequence and the bivalent aptamer sequence, the sequence inserted with 2G bases significantly stimulated ThT fluorescence; The number of inserted bases in the appropri...
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