Bioinformatics designing of 10-23 deoxyribozyme against noncoding region before start codon of beta-galactosidase gene (lacZ) in pGEM-T vector

Background and aims: Deoxyribozymes are oligoribodeoxynucleotides that catalyze reactions such as cutting RNA and have diagnostic and therapeutic applications. Deoxyribozyme 10-23 includes a catalytic domain dependent on a fixed 15-nucleotic (mer) cation and two variable binding arms that cause the specificity of enzymes. Lactose operon is used in the white-blue screening process. This operon includes three polycistronic genes. In this study, a deoxyribozyme against α-peptide beta-galactosidase gene in the lactose operon was designed. Methods: pGEM-T map was obtained from addgene server and α-peptide gene sequence was determined. Then, using expasy website proper protein frame in comparison with various reading frames was determined. In this step, whole sequence was reversed and mRNA sequence was achieved. Secondary structure with the lowest free energy was gained using mfold server. Considering the fact that 10-23 deoxyribozyme has cutting capability between a unpaired purine and pairs pyrimidine; an AC was selected in ribosome binding site in the untranslated region and then 9 open bases on either side of it was used as a binding arms. Investigation of the absence of similar sequences in host bacteria was performed by NCBI server. Finally, activity and binding of deoxyribozyme was predicted by the mfold server. Results: The results of this study showed that the designed deoxyribozyme had a relatively high Tm with two 9-nucleotide arms, which increased its effectiveness. Conclusion: The results of this study can be used to control the expression of lacZ gene as a biomarker

Evaluating the effects of mono and disaccharids on gold nanoparticles used for biosensors

Background and Aims: Recently,­ in addition to numerous molecular methods available for diagnosis, methods based on the design of various biosensors are widely used. Some of these biosensors are Lateral flow tests based on gold nanoparticles. Considering the importance of the application of gold nanoparticles as markers in biosensors and also the need for the use of sugars in the construction of biosensors,this study was proposed to investigate the effects of various sugars (monosaccharides and disaccharides) on gold nanoparticles for the development of diagnostic biosensors. Methods: In this experimental study, after synthesis of gold nanoparticles chemically, by size, measuring maximum absorption at 520 nm, and concentration of nanoparticles a spectrophotometer and DLS method were used. Then, considering the importance of using sugars in the manufacture of biosensors, a gradient of 5 to 500 mM glucose, fructose, sucrose, and trehalose, and their absorption spectra, after exposure to gold nanoparticles at 450 to 700 nm were utilized. Results: The results of the spectrophotometric spectrum of synthesized nanoparticles in the presence and absence of various sugars of monosaccharide and disaccharide, indicates a decrease in the maximum absorption of nanoparticles after the application of a steady slope of sugar types on them. Slope of glucose, sucrose and trehalose sugars has no effect on the accumulation and aggregation of gold nanoparticles, while fructose at concentrations up to 50 mM leads to the accumulation of gold nanoparticles. Conclusion: Due to the necessity of using sugars in the production of bionanosensors, it seems that the use of ketone sugars, such as fructose, at concentrations of 50 mM upwards is not suitable for making biosensors

Lanthanide Cofactors Accelerate DNA-Catalyzed Synthesis of Branched RNA

Most deoxyribozymes (DNA catalysts) require metal ions as cofactors for catalytic activity, with Mg²⁺, Mn²⁺, and Zn²⁺ being the most represented activators. Trivalent transition-metal ions have been less frequently considered. Rare earth ions offer attractive properties for studying metal ion binding by biochemical and spectroscopic methods. Here we report the effect of lanthanide cofactors, in particular terbium (Tb³⁺), for DNA-catalyzed synthesis of 2′,5′-branched RNA. We found up to 10⁴-fold increased ligation rates for the 9F7 deoxribozyme using 100 μM Tb³⁺ and 7 mM Mg²⁺, compared to performing the reaction with 7 mM Mg²⁺ alone. Combinatorial mutation interference analysis (CoMA) was used to identify nucleotides in the catalytic region of 9F7 that are essential for ligation activity with different metal ion combinations. A minimized version of the DNA enzyme sustained high levels of Tb³⁺-assisted activity. Sensitized luminescence of Tb³⁺ bound to DNA in combination with DMS probing and DNase I footprinting results supported the CoMA data. The accelerating effect of Tb³⁺ was confirmed for related RNA-ligating deoxyribozymes, pointing toward favorable activation of internal 2′-OH nucleophiles. The results of this study offer fundamental insights into nucleotide requirements for DNA-catalyzed RNA ligation and will be beneficial for practical applications that utilize 2′,5′-branched RNA

Bioinformatics Designing of 10-23 Deoxyribozyme against Coding Region of Beta-galactosidase Gene

Background: Deoxyribozymes (Dzs) can play a role as gene expression inhibitors at mRNA level. Among Dzs, the 10-23 deoxyribozyme has significant potentials for treatment of diseases. Designed Dz includes a catalytic core made of 15 deoxyribonucleotides and two binding arms consisted of 6-12 nucleotides for site specific binding to target RNA and hydrolysis. The enzyme has characteristic features for cleavage of the RNA target between an unpaired purine (A, G) and a paired pyrimidine (C or U). In this study, 10-23 Dz is designed for the coding region of the α-peptide of a lacZ gene. Material and Methods: The primary sequence of a plasmid with α-complementation ability was taken from addgene database. To confirm sequence validity, ExPASy was used to analyze related ORFs for the retrieved sequence. The ORF with identical sequence to α-peptide was selected in the reverse complement sequence. Subsequently, the secondary structure of the α-peptide was analyzed in DINAMelt web server and Mfold software. Then the intended target site was selected inside the coding region of the α-peptide. The Dzs sequence was designed for the target site with nucleotide binding arms. Results and conclusion: The resulted Dz in this study can be used as a promising catalytic DNA inside bacterial cells for blue-white screening. Criteria such as biological stability and catalytic rate of such enzymes must be evaluated in vivo and in vitro

Site-Specific Labeling of RNA at Internal Ribose Hydroxyl Groups: Terbium-Assisted Deoxyribozymes at Work

A general and efficient single-step method was established for site-specific post-transcriptional labeling of RNA. Using Tb³⁺ as accelerating cofactor for deoxyribozymes, various labeled guanosines were site-specifically attached to 2′-OH groups of internal adenosines in <i>in vitro</i> transcribed RNA. The DNA-catalyzed 2′,5′-phosphodiester bond formation proceeded efficiently with fluorescent, spin-labeled, biotinylated, or cross-linker-modified guanosine triphosphates. The sequence context of the labeling site was systematically analyzed by mutating the nucleotides flanking the targeted adenosine. Labeling of adenosines in a purine-rich environment showed the fastest reactions and highest yields. Overall, practically useful yields >70% were obtained for 13 out of 16 possible nucleotide (nt) combinations. Using this approach, we demonstrate preparative labeling under mild conditions for up to ∼160-nt-long RNAs, including spliceosomal U6 small nuclear RNA and a cyclic-di-AMP binding riboswitch RNA