RNA therapeutics inhibit the expression of specific proteins/RNAs by targeting complementary sequences of corresponding genes or encode proteins for the synthesis desired genes to treat genetic diseases. RNA-based therapeutics are categorized as oligonucleotide drugs (antisense oligonucleotides, small interfering RNA, RNA aptamers), and mRNA drugs. The antisense oligonucleotides and small interfering RNA for treatment of genetic diseases have been approved by the FDA in the United States, while RNA aptamers and mRNA drugs are still in clinical trials. Chemical modifications can be applied to RNA drugs, such as pseudouridine modification of mRNA, to reduce immunogenicity and improve the efficacy. The secure and effective delivery systems such as lipid-based nanoparticles, extracellular vesicles, and virus-like particles are under development to address stability, specificity, and safety issues of RNA drugs. This article provides an overview of the specific molecular mechanisms of eleven RNA drugs currently used for treating genetic diseases, and discusses the research progress of chemical modifications and delivery systems of RNA drugs.
Aptamers, Nucleotide ; RNA, Small Interfering/therapeutic use* ; RNA, Messenger ; Oligonucleotides, Antisense/therapeutic use*

The computer information technology that has penetrated into every aspect of our lives, can not only assist the screening of drugs, but also simulate the effect of drugs. At present, computer-aided technologies have been used to screen aptamers, which play an important role in improving the screening efficiency and screening high affinity binding aptamers. This review summarized the screening methods of aptamers through computer-aided sequence evaluation, structural analysis and molecular docking.
Aptamers, Nucleotide ; Computers ; Molecular Docking Simulation ; SELEX Aptamer Technique/methods*

Nucleic acid aptamers, broad-spectrum target-specific single-stranded oligonucleotides, serve as molecules in targeted therapy, targeted delivery and disease diagnosis for the treatment of tumor or microbial infection and clinical detection. Due to the existence of components in the use of traditional Chinese medicine(TCM), the target is difficult to concentrate and the specificity of treatment is poor. The effective components of TCM are toxic components, so a highly sensitive detection method is urgently needed to reduce the toxicity problem at the same time. The combined application of TCM and modern medical treatment strategy are difficult and cannot improve the therapeutic effect. Aptamers, advantageous in biosensors, aptamer-nanoparticles for targeted drug delivery, and aptamer-siRNA chimeras, are expected to connect Chinese medicinals with nanotechnology, diagnostic technology and combined therapies. We summarized the preparation, screening, and modification techniques of nucleic acid aptamers and the biomedical applications and advantages in therapy, targeting, and diagnosis, aiming at providing a reference for the in-depth research and development in TCM.
Aptamers, Nucleotide ; Drug Delivery Systems ; Medicine, Chinese Traditional ; Nucleic Acids ; RNA, Small Interfering

Mixed lineage leukemia proteins (MLL) are the key histone lysine methyltransferases that regulate expression of diverse genes. Aberrant activation of MLL promotes leukemia as well as solid tumors in humans, highlighting the urgent need for the development of an MLL inhibitor. We screened and isolated MLL1-binding ssRNAs using SELEX (Systemic Evolution of Ligands by Exponential enrichment) technology. When sequences in sub-libraries were obtained using next-generation sequencing (NGS), the most enriched aptamers—APT1 and APT2—represented about 30% and 26% of sub-library populations, respectively. Motif analysis of the top 50 sequences provided a highly conserved sequence: 5′-A[A/C][C/G][G/U][U/A]ACAGAGGG[U/A]GG[A/C] GAGUGGGU-3′. APT1, APT2, and APT5 embracing this motif generated secondary structures with similar topological characteristics. We found that APT1 and APT2 have a good binding activity and the analysis using mutated aptamer variants showed that the site information in the central region was critical for binding. In vitro enzyme activity assay showed that APT1 and APT2 had MLL1 inhibitory activity. Three-dimensional structure prediction of APT1-MLL1 complex indicates multiple weak interactions formed between MLL1 SET domain and APT1. Our study confirmed that NGS-assisted SELEX is an efficient tool for aptamer screening and that aptamers could be useful in diagnosis and treatment of MLL1-mediated diseases.
Aptamers, Nucleotide ; Conserved Sequence ; Diagnosis ; Histones ; Humans ; In Vitro Techniques ; Leukemia ; Ligands ; Lysine ; Mass Screening ; Methyltransferases ; Myeloid-Lymphoid Leukemia Protein ; RNA

OBJECTIVETo develop a novel colorimetric method for detecting the tumor biomarker vascular endothelial growth factor (VEGF) based on aptamer and magnetic beads.

METHODSThe capture aptamer was hybridized to urease functionalized single-stranded DNA (ssDNA) and immobilize on the surface of magnetic beads by specific biotin-avidin binding. In the presence of VEGF, aptamers bound to VEGF to form a specific stem-loop structure to release the urease functionalized ssDNA. After separation, the supernatant was transferred to a tube and urea and phenol red were added. Urease hydrolyzed urea to produce ammonia to cause an increase of the pH value and a color change of phenol red. The results were inspected with either the naked eyes or by a UV spectrophotometer.

RESULTSUnder optimized conditions, the detection system showed a good linear relationship for VEGF detection in the range of 0.1 to 10 pmol/L with a detection limit as low as 0.06 pmol/L. The results of VEGF detection in the serum of patients with lung cancer were consistent with those using an ELISA Kit. The results of examination of 10 serum samples with this aptamer-based method and ELISA kit showed that the accuracy of this method was 90%.

CONCLUSIONThis aptamer-based system provides an simple and convenient method for VEGF detection with a high sensitivity and selectivity.

Aptamers, Nucleotide ; Biomarkers, Tumor ; Colorimetry ; DNA, Single-Stranded ; Humans ; Lung Neoplasms ; Nucleic Acid Hybridization ; Vascular Endothelial Growth Factor A ; analysis

RNA-binding protein exerts important biological function by specifically recognizing RNA motif. SELEX (Systematic evolution of ligands by exponential enrichment), an in vitro selection method, can obtain consensus motif with high-affinity and specificity for many target molecules from DNA or RNA libraries. Here, we combined SELEX with next-generation sequencing to study the protein-RNA interaction in vitro. A pool of RNAs with 20 bp random sequences were transcribed by T7 promoter, and target protein was inserted into plasmid containing SBP-tag, which can be captured by streptavidin beads. Through only one cycle, the specific RNA motif can be obtained, which dramatically improved the selection efficiency. Using this method, we found that human hnRNP A1 RRMs domain (UP1 domain) bound RNA motifs containing AGG and AG sequences. The EMSA experiment indicated that hnRNP A1 RRMs could bind the obtained RNA motif. Taken together, this method provides a rapid and effective method to study the RNA binding specificity of proteins.
Aptamers, Nucleotide ; Gene Library ; Heterogeneous Nuclear Ribonucleoprotein A1 ; genetics ; High-Throughput Nucleotide Sequencing ; Humans ; RNA ; SELEX Aptamer Technique

Antibodies, Monoclonal, Humanized ; therapeutic use ; Aptamers, Nucleotide ; therapeutic use ; Bevacizumab ; Humans ; Macular Edema ; drug therapy ; Ranibizumab ; Receptors, Vascular Endothelial Growth Factor ; therapeutic use ; Recombinant Fusion Proteins ; therapeutic use ; Retinal Vein Occlusion ; drug therapy ; Vascular Endothelial Growth Factor A ; antagonists & inhibitors ; Visual Acuity ; drug effects

OBJECTIVEThis review aims to summarize the progress of current clinical studies in ocular angiogenesis treated with anti-vascular endothelial growth factor (VEGF) therapy and to discuss the benefits and challenges of the treatment.

DATA SOURCESPubmed, Embase and the Cochrane Library were searched with no limitations of language and year of publication.

STUDY SELECTIONClinical trials and case studies presented at medical conferences and published in peer-reviewed literature in the past decade were reviewed.

RESULTSAnti-VEGF agents have manifested great potential and promising outcomes in treating ocular neovascularization, though some of them are still used as off-label drugs. Intravitreal injection of anti-VEGF agents could be accompanied by devastating ocular or systemic complications, and intimate monitoring in both adult and pediatric population are warranted. Future directions should be focused on carrying out more well-designed large-scale controlled trials, promoting sustained duration of action, developing safer and more efficient generation of anti-VEGF agents.

CONCLUSIONSAnti-VEGF treatment has proved to be beneficial in treating both anterior and posterior neovascular ocular diseases. However, more safer and affordable antiangiogenic agencies and regimens are warranted to be explored.

Antibodies, Monoclonal, Humanized ; therapeutic use ; Aptamers, Nucleotide ; therapeutic use ; Bevacizumab ; Eye ; blood supply ; drug effects ; pathology ; Humans ; Neovascularization, Pathologic ; drug therapy ; Ranibizumab ; Vascular Endothelial Growth Factor A ; antagonists & inhibitors

Aptamers are single-stranded oligonucleotide ligands selected from a random pool of oligonucleotide sequences by systematic evolution of ligands by exponential enrichment. With the advantages of binding to targets with high specificity and affinity, aptamers have been used in the diagnosis widely, such as biochemistry detection, discovery of new tumor markers, molecular imaging, pathogenic and microorganism detection, showing a good application prospect.
Aptamers, Nucleotide ; Diagnosis ; Disease ; Humans ; Sensitivity and Specificity

OBJECTIVE: To select specific DNA aptamer for determining ketamine by FluMag-SELEX. METHODS: Based on magnetic beads with tosyl surface modification as solid carrier and ketamine as target, a random ssDNA library with total length of 78 bp in vitro was compounded. After 13 rounds screening, DNA cloning and sequencing were done. Primary and secondary, structures were analyzed. The affinity, specificity and Kd values of selected aptamer were measured by monitoring the fluorescence intensity. RESULTS: Two ssDNA aptamers (Apt#4 and Apt#8) were successfully selected with high and specific abilities to bind ketamine as target with Kd value of 0.59 and 0.66 μmol/L. The prediction of secondary structure was main stem-loop and G-tetramer. The stem was the basis of stability of aptamer's structure. And loop and G-tetramer was the key of specific binding of ketamine. CONCLUSION FluMag-SELEX can greatly improve the selection efficiency of the aptamer, obtain the ketamine-binding DNA aptamer, and develop a new method for rapid detection of ketamine.
Aptamers, Nucleotide/metabolism* ; DNA ; DNA, Single-Stranded/genetics* ; In Vitro Techniques ; Ketamine/metabolism* ; Oligonucleotides ; SELEX Aptamer Technique/methods*