Construction Of DNA-Based Photochemical Biosensors And Their Researches In MRNA Detection

Messenger RNA(mRNA)plays an important role in various life processes.Achieving accurate detection of low-abundance mRNA not only helps us better understand the physiological functions of mRNA,but also provides important information for disease diagnosis and treatment.However,it is still challenging to develop more sensitive,faster and more accurate mRNA detection methods due to the inherent feature of sequence similarity between homologous mRNA.In order to realize the sensitive and accurate detection of TK1 mRNA,two kinds of photochemical biosensors using DNA as the recognition element to respond to thymus kinase 1(TK1)mRNA were constructed in this paper.The main contents are as follows:1.A phosphorescence resonance energy transfer(PRET)-based sensor was constructed to achieve accurate detection of TK1 mRNA in biological samples.Firstly,persistent luminescent nanoparticles(PL)were synthesized by direct aqueous method and surface aminated.It was then coupled with carboxyl-modified DNA(I-DNA)to obtain I-DNA strand-modified PL(PL-DNA).Afterwards,polydopamine(PDA)was synthesized,and the hairpin DNA(H)and the substrate strand(S)were adsorbed by electrostatic and π-π stacking interactions.The PL-DNA was then hybridized with the S strand to prepare a phosphorescent biosensor.In the absence of the target TK1 mRNA,the distance between the PL at the end of the PL-DNA hybrid and the surface of the PDA was about 7.48 nm because the number of bases in the duplex of the PL-DNA hybrid was 22.Therefore,PRET occurred between PL and PDA,resulting in the quenching of the phosphorescence of PL by PDA.The stem of the hairpin H was complementary to the target,and the H was opened by the target to form a flexible end.The opened end walked along the PDA track to the similar SI duplex to undergo a strand displacement reaction,which displaced the signal strand from the substrate strand,resulting in the closure of PRET and the recovery of phosphorescence.The response of this sensor to TK1 mRNA was linear in the range of 0-200 n M with a detection limit of 1.74 n M.In addition,the sensor effectively avoided the interference of autofluorescence in complex biological samples,enabling sensitive and accurate detection of TK1 mRNA in serum samples.2.A fluorescent biosensor based on proximity catalytic hairpin assembly(CHA)was constructed to achieve sensitive detection of TK1 mRNA.First,amino-modified magnetic nanoparticles(N-MNP)were synthesized and coupled with carboxyl-modified substrate strands(S-DNA)to obtain S-DNA strand-modified magnetic probes(MP).DNA Walker was obtained by hybridizing S-DNA with hairpin H1(modified Cy3 and BHQ2)and H2 ends;based on the partial characteristic sequence of TK1 mRNA,the walking arms(W1,W2 and antisense strand L)in response to TK1 mRNA were designed.These two parts together constitute a fluorescent biosensor.When no target was present,the walking arm was locked by the L chain and the adjacent CHA reaction is not driven,At this time,the fluorescence of Cy3 was quenched.Once the target was present,L was replaced and the DNA arms were opened.The opened walking arms moved freely along the three-dimensional orbit,inducing the opening of the hairpin H1 and the recovery of Cy3 fluorescence.The opened H1 portion could in turn hybridize with H2 to form a double strand,releasing the walking arm for the next walk.The bipedal walker achieved sensitive detection of TK1 mRNA with a detection limit as low as 14 p M,and exhibited excellent selectivity between homologous and mismatched mRNA.3.A Y-type fluorescent probe based on G-quadruplex and fluorescence resonance energy transfer strategy realizes the spatially ordered detection of Survivin mRNA.In this work,a Y-type fluorescent probe(Y-probe)for spatially ordered detection of Survivin mRNA was constructed by incubation of six DNA single strands at 37 o C.In the absence of the target,the G-quadruplex structure cannot be formed and thus Thioflavin T(Th T)does not emit light.And the dye TAMRA has a certain intensity of fluorescence.In the presence of the long Survivin mRNA sequence,the three recognition strands in the Y-probe are replaced by the target to form a split G-quadruplex and a fluorescence resonance energy transfer pair.At this point split G-quadruplexes are formed and TAMRA fluorescence is quenched.This signal occurs only when the target sequence is completely specific,enabling spatially ordered detection of mRNA.On this basis,the detection of the sequence of Survivin mRNA inconsistent with the probe was compared,and it was proved that the probe could accurately identify the sequence of Survivin mRNA.A detection limit of 17.53 n M was also obtained.