Short RNA Research Articles

Nucleoprotein phase-separation affinities revealed via atomistic simulations of short peptide and RNA fragments.

Liquid-liquid phase separation of proteins and nucleic acids into condensate phases is a versatile mechanism for ensuring compartmentalization of cellular biochemistry. RNA molecules play critical roles in these condensates, particularly in transcriptional regulation and stress responses, exhibiting a wide range of thermodynamic and dynamic behaviors. However, deciphering the molecular grammar that governs the stability and dynamics of protein-RNA condensates remains challenging due to the multicomponent and heterogeneous nature of these biomolecular mixtures. In this study, we employ atomistic simulations of twenty distinct mixtures containing minimal RNA and peptide fragments to dissect the phase-separating affinities of all twenty amino acids in the presence of RNA. Our findings elucidate chemically specific interactions, hydration profiles, and ionic effects that synergistically promote or suppress protein-RNA phase separation. We map a ternary phase diagram of interactions, identifying four distinct groups of residues that promote, maintain, suppress, or disrupt protein-RNA clusters.

Contrasting and combining transcriptome complexity captured by short and long RNA sequencing reads.

Mapping transcriptomic variations using either short- or long-reads RNA sequencing is a staple of genomic research. Long reads are able to capture entire isoforms and overcome repetitive regions, while short reads still provide improved coverage and error rates. Yet how to quantitatively compare the technologies, can we combine those, and what may be the benefit of such a combined view remain open questions. We tackle these questions by first creating a pipeline to assess matched long and short reads data using a variety of transcriptome statistics. We find that across datasets, algorithms, and technologies, matched short reads data detects roughly 30% more splice junctions such that 10-30% of the splice junctions included at 20% or more by short reads are missed by long reads. In contrast, long reads detect many more intron retention events and can detect full isoforms, pointing to the benefit of combining the technologies. We introduce MAJIQ-L, an extension of the MAJIQ software to enable a unified view of transcriptome variations from both technologies and demonstrate its benefits. Our software can be used to assess any future long-read technology or algorithm, and combine it with short reads data for improved transcriptome analysis.

Acquired sensorineural hearing loss, oxidative stress, and microRNAs.

Hearing loss is the third leading cause of human disability. Age-related hearing loss, one type of acquired sensorineural hearing loss, is largely responsible for this escalating global health burden. Noise-induced, ototoxic, and idiopathic sudden sensorineural are other less common types of acquired hearing loss. The etiology of these conditions is complex and multi-factorial involving an interplay of genetic and environmental factors. Oxidative stress has recently been proposed as a likely linking cause in most types of acquired sensorineural hearing loss. Short non-coding RNA sequences known as microRNAs (miRNAs) have increasingly been shown to play a role in cellular hypoxia and oxidative stress responses including promoting an apoptotic response. Sensory hair cell death is a central histopathological finding in sensorineural hearing loss. As these cells do not regenerate in humans, it underlies the irreversibility of human age-related hearing loss. Ovid EMBASE, Ovid MEDLINE, Web of Science Core Collection, and ClinicalTrials. gov databases over the period August 1, 2018 to July 31, 2023 were searched with "hearing loss," "hypoxamiRs," "hypoxia," "microRNAs," "ischemia," and "oxidative stress" text words for English language primary study publications or registered clinical trials. Registered clinical trials known to the senior author were also assessed. A total of 222 studies were thus identified. After excluding duplicates, editorials, retractions, secondary research studies, and non-English language articles, 39 primary studies and clinical trials underwent full-text screening. This resulted in 11 animal, in vitro, and/or human subject journal articles and 8 registered clinical trial database entries which form the basis of this narrative review. MiRNAs miR-34a and miR-29b levels increase with age in mice. These miRNAs were demonstrated in human neuroblastoma and murine cochlear cell lines to target Sirtuin 1/peroxisome proliferator-activated receptor gamma coactivator-1-alpha (SIRT1/PGC-1α), SIRT1/p53, and SIRT1/hypoxia-inducible factor 1-alpha signaling pathways resulting in increased apoptosis. Furthermore, hypoxia and oxidative stress had a similar adverse apoptotic effect, which was inhibited by resveratrol and a myocardial inhibitor-associated transcript, a miR-29b competing endogenous mRNA. Gentamicin reduced miR-182-5p levels and increased cochlear oxidative stress and cell death in mice - an effect that was corrected by inner ear stem cell-derived exosomes. There is ongoing work seeking to determine if these findings can be effectively translated to humans.

Short Repeat Ribonucleic Acid Reduces Cytotoxicity by Preventing the Aggregation of TDP-43 and Its 25 KDa Carboxy-Terminal Fragment.

TAR DNA/RNA-binding protein 43 kDa (TDP-43) proteinopathy is a hallmark of neurodegenerative disorders, such as amyotrophic lateral sclerosis, in which cytoplasmic aggregates containing TDP-43 and its C-terminal fragments, such as TDP-25, are observed in degenerative neuronal cells. However, few reports have focused on small molecules that can reduce their aggregation and cytotoxicity. Here, we show that short RNA repeats of GGGGCC and AAAAUU are aggregation suppressors of TDP-43 and TDP-25. TDP-25 interacts with these RNAs, as well as TDP-43, despite the lack of major RNA-recognition motifs using fluorescence cross-correlation spectroscopy. Expression of these RNAs significantly decreases the number of cells harboring cytoplasmic aggregates of TDP-43 and TDP-25 and ameliorates cell death by TDP-25 and mislocalized TDP-43 without altering the cellular transcriptome of molecular chaperones. Consequently, short RNA repeats of GGGGCC and AAAAUU can maintain proteostasis by preventing the aggregation of TDP-43 and TDP-25.

The plasma miRNAome in ADNI: Signatures to aid the detection of at-risk individuals.

MicroRNAs are short non-coding RNAs that control proteostasis at the systems level and are emerging as potential prognostic and diagnostic biomarkers for Alzheimer's disease (AD). We performed small RNA sequencing on plasma samples from 847 Alzheimer's Disease Neuroimaging Initiative (ADNI) participants. We identified microRNA signatures that correlate with AD diagnoses and help predict the conversion from mild cognitive impairment (MCI) to AD. Our data demonstrate that plasma microRNA signatures can be used to not only diagnose MCI, but also, critically, predict the conversion from MCI to AD. Moreover, combined with neuropsychological testing, plasma microRNAome evaluation helps predict MCI to AD conversion. These findings are of considerable public interest because they provide a path toward reducing indiscriminate utilization of costly and invasive testing by defining the at-risk segment of the aging population. We provide the first analysis of the plasma microRNAome for the ADNI study. The levels of several microRNAs can be used as biomarkers for the prediction of conversion from MCI to AD. Adding the evaluation of plasma microRNA levels to neuropsychological testing in a clinical setting increases the accuracy of MCI to AD conversion prediction.

Differential miRNA Expression Patterns between TallyHo/JngJ Mice and Non-Diabetic SWR/J Mice

Abstract Type 2 diabetes mellitus (T2DM) increases the susceptibility of bone fragility. The underlying mechanisms have, however, remained largely unknown. MicroRNAs (miRNAs) are short single-stranded non-coding RNA molecules with utility as biomarkers due to their easy accessibility and stability in bodily fluids. Here, we aimed to use an unbiased approach to identify miRNAs dysregulated in a polygenic mouse model of T2DM. Genome-wide analysis of miRNAs in serum, bone marrow and bone from the polygenic TallyHo/JngJ (TH) mice, which recapitulate T2DM in humans, was performed. This analysis was compared to the recommended control SWR/J and a strain-matched non-diabetic control (TH-ND). When comparing TH mice with TH-ND using an adjusted P-value (FDR) cut-off of 0.2 to identify differentially expressed miRNAs, mmu-miR-466i-5p and mmu-miR-1195 were found to be up-regulated in both serum and in bone marrow. Dysregulated miRNAs were not found in bone tissue. When comparing TH-ND mice with SWR/J using the same FDR cut-off, mmu-miR-351-5p and mmu-miR-322-3p were upregulated in both bone marrow and serum, while mmu-miR-449a-5p and mmu-miR-6240 were downregulated in bone marrow and serum. Dysregulated miRNAs in bone marrow or cortical bone compared to serum between TH-ND mice and SWR/J were investigated for their cell-type enrichment to identify putative donor cells and their gene target networks. Gene target network analysis revealed genes involved in diabetes-related signaling pathways as well as in diabetic bone disease. Cell-type enrichment analysis identified hsa-miR-449a enriched in immune cells, hsa-miR-592 in hepatocytes and endothelial cells, while hsa-miR-424-3p, hsa-miR-1-3p and hsa-miR-196b-5p were enriched in mesenchymal stem cells and their derived tissues. In conclusion, our comparative miRNA profiling sheds light on differential expression patterns between SWR/J and both subgroups of TH. No differences were observed between TH and TH-ND suggesting the genetical background of SWR/J may be responsible for the change of dysregulated miRNA.

Thg1 family 3'-5' RNA polymerases as tools for targeted RNA synthesis.

Members of the 3'-5' RNA polymerase family, comprised of tRNAHis guanylyltransferase (Thg1) and Thg1-like proteins (TLPs), catalyze templated synthesis of RNA in the reverse direction to all other known 5'-3' RNA and DNA polymerases. The discovery of enzymes capable of this reaction raised the possibility of exploiting 3'-5' polymerases for posttranscriptional incorporation of nucleotides to the 5'-end of nucleic acids without ligation, and instead by templated polymerase addition. To date, studies of these enzymes have focused on nucleotide addition to highly structured RNAs, such as tRNA and other noncoding RNAs. Consequently, general principles of RNA substrate recognition and nucleotide preferences that might enable broader application of 3'-5' polymerases have not been elucidated. Here, we investigated the feasibility of using Thg1 or TLPs for multiple nucleotide incorporation to the 5'-end of a short duplex RNA substrate, using a templating RNA oligonucleotide provided in trans to guide 5'-end addition of specific sequences. Using optimized assay conditions, we demonstrated a remarkable capacity of certain TLPs to accommodate short RNA substrate-template duplexes of varying lengths with significantly high affinity, resulting in the ability to incorporate a desired nucleotide sequence of up to eight bases to 5'-ends of the model RNA substrates in a template-dependent manner. This work has further advanced our goals to develop this atypical enzyme family as a versatile nucleic acid 5'-end labeling tool.

Advancing microRNA target site prediction with transformer and base-pairing patterns.

MicroRNAs (miRNAs) are short non-coding RNAs involved in various cellular processes, playing a crucial role in gene regulation. Identifying miRNA targets remains a central challenge and is pivotal for elucidating the complex gene regulatory networks. Traditional computational approaches have predominantly focused on identifying miRNA targets through perfect Watson-Crick base pairings within the seed region, referred to as canonical sites. However, emerging evidence suggests that perfect seed matches are not a prerequisite for miRNA-mediated regulation, underscoring the importance of also recognizing imperfect, or non-canonical, sites. To address this challenge, we propose Mimosa, a new computational approach that employs the Transformer framework to enhance the prediction of miRNA targets. Mimosa distinguishes itself by integrating contextual, positional and base-pairing information to capture in-depth attributes, thereby improving its predictive capabilities. Its unique ability to identify non-canonical base-pairing patterns makes Mimosa a standout model, reducing the reliance on pre-selecting candidate targets. Mimosa achieves superior performance in gene-level predictions and also shows impressive performance in site-level predictions across various non-human species through extensive benchmarking tests. To facilitate research efforts in miRNA targeting, we have developed an easy-to-use web server for comprehensive end-to-end predictions, which is publicly available at http://monash.bioweb.cloud.edu.au/Mimosa.

MicroRNAs: pioneering regulators in Alzheimer’s disease pathogenesis, diagnosis, and therapy

This article delves into Alzheimer’s disease (AD), a prevalent neurodegenerative condition primarily affecting the elderly. It is characterized by progressive memory and cognitive impairments, severely disrupting daily life. Recent research highlights the potential involvement of microRNAs in the pathogenesis of AD. MicroRNAs (MiRNAs), short non-coding RNAs comprising 20–24 nucleotides, significantly influence gene regulation by hindering translation or promoting degradation of target genes. This review explores the role of specific miRNAs in AD progression, focusing on their impact on β-amyloid (Aβ) peptide accumulation, intracellular aggregation of hyperphosphorylated tau proteins, mitochondrial dysfunction, neuroinflammation, oxidative stress, and the expression of the APOE4 gene. Our insights contribute to understanding AD’s pathology, offering new avenues for identifying diagnostic markers and developing novel therapeutic targets.

Advances in the study of the mechanism of action of miR‑22 in liver lesions (Review).

Globally, nearly 2 million deaths annually are attributed to the development of liver diseases, with liver cancer and cirrhosis being particularly prominent, which makes liver disease a significant global health concern. Cirrhosis is closely linked to the evolution of hepatitis, hepatic fibrosis and fatty liver. However, most liver diseases have an insidious onset, are challenging to treat and the prognosis and efficacy of current therapies are unsatisfactory, which can result in irreversible functional damage to the liver. Therefore, there is an urgent need to explore the molecular mechanisms underlying liver disease and identify new biomarkers and therapeutic targets. In previous years, microRNAs (miRs), a class of short non-coding RNAs comprising 17-25 nucleotides, have attracted attention for their roles in various types of liver diseases. Among them, miR-22 serves a unique role in mediating multiple pathway mechanisms and epigenetic modifications and can act both as an inhibitor of liver cancer and a metabolic blocker. Given its close association with the liver, several studies have reported that the differential expression of miR-22 regulates the metabolic process of liver cancer and is involved in the evolution of hepatic fibrosis and steatohepatitis, making it a potential target for early diagnosis and treatment. The present manuscript aimed to comprehensively review the key role of miR-22 in the evolution of liver diseases and offer valuable references and guidance for subsequent studies by identifying its specific mechanism of action and future development prospects.

Serum tsncRNAs reveals novel potential therapeutic targets of Salvianolic Acid B on atherosclerosis

BackgroundSalvianolic Acid B (SalB) has been proven to delay the progression of atherosclerosis. The therapeutic mechanisms of this compound are unclear. A novel class of short non-coding RNAs, pre-transfer RNA and mature transfer RNA (tsncRNAs) may regulate gene expression. TsncRNAs-sequencing revealed novel therapeutic targets for SalB. This is the first study focusing on tsncRNAs to treat atherosclerosis using SalB. PurposeTo explore the potential mechanism of SalB treating atherosclerosis through tsncRNAs. MethodsFive groups of mice were created at random: control group (CON), atherosclerosis model group (MOD), SalB with high dose-treated group (SABH), SalB with low dose-treated group (SABL), and Simvastatin-treated group (ST). Aortic sinus plaque, body weight and inflammatory cytokines were evaluated. The Illumina NextSeq equipment was used to do expression profiling of tsncRNAs from serum. The targets of tsncRNAs were then predicted using tRNAscan and TargetScan. The KEGG pathway and GO analysis were utilized to forecast the bioinformatics analysis. Potential tsncRNAs and associated mRNAs were validated using quantitative real-time PCR. ResultstRF-Glu-CTC-014 and tRF-Gly-GCC-074 were markedly increased by SalB with high dose treatment and validated with quantitative real-time PCR. Two mRNAs SRF and Arrb related to tRF-Glu-CTC-014 changed consistently. GO analysis revealed that the altered target genes of the selected tsncRNAs were most enriched in protein binding and cellular process. Moreover, KEGG pathway analysis demonstrated that altered target genes of tsncRNAs were most enriched in MAPK signaling pathway. ConclusionSalB can promote the expression of tRF-Glu-CTC-014 to treat atherosclerosis.

Gene expression analysis of microRNA-1285 in the South Indian oral squamous cell carcinoma population.

Oral squamous cell carcinoma (OSCC) is the most common oral malignant tumor, which has poor prognosis. The traditional investigative modality is invasive biopsy which is the gold standard for diagnosis. In recent years, alternative methods like non-invasive biomarkers have been studied for their potential role in early diagnosis and prognosis. Among them, microRNAs (miRNAs or miRs) are short non-coding RNAs that regulate gene expression in various diseases, including OSCC. Several miRNAs are being researched as non-invasive biomarkers as well as novel therapeutic targets in the treatment of OSCC. MiR expression can be upregulated or downregulated in OSCC. Among the reported miRNAs, miR-1285 is an important miRNA found to be involved in OSCC. The aim of the current study was to quantify the levels of miR-1285 in OSCC samples and to validate their potential role as biomarkers for OSCC detection. Sixteen samples of cancer tissue and normal tissue were evaluated from a total of 25 patients, in the study, conducted in the Department of Oral and Maxillofacial Surgery. The tissues were processed for H&E staining and gene expression analysis of miR-1285. The samples were collected after proper informed consent from the patients. Total RNA isolated was reverse transcribed into cDNA which was used in the gene expression analysis using qRT-PCR. The histopathological examination confirmed the OSCC cases and the gene expression analysis revealed that miR-1285 was significantly downregulated in OSCC tissues. Since miR-1285 showed significant difference between the OSCC and normal tissues it could be postulated as a potential biomarker and therapeutic target for OSCC. Further in-vitro and in-vivo studies could validate their functional role in OSCC.

A resource of identified and annotated lincRNAs expressed during somatic embryogenesis development in Norway spruce.

Long non-coding RNAs (lncRNAs) have emerged as important regulators of many biological processes, although their regulatory roles remain poorly characterized in woody plants, especially in gymnosperms. A major challenge of working with lncRNAs is to assign functional annotations, since they have a low coding potential and low cross-species conservation. We utilised an existing RNA-Sequencing resource and performed short RNA sequencing of somatic embryogenesis developmental stages in Norway spruce (Picea abies L. Karst). We implemented a pipeline to identify lncRNAs located within the intergenic space (lincRNAs) and generated a co-expression network including protein coding, lincRNA and miRNA genes. To assign putative functional annotation, we employed a guilt-by-association approach using the co-expression network and integrated these results with annotation assigned using semantic similarity and co-expression. Moreover, we evaluated the relationship between lincRNAs and miRNAs, and identified which lincRNAs are conserved in other species. We identified lincRNAs with clear evidence of differential expression during somatic embryogenesis and used network connectivity to identify those with the greatest regulatory potential. This work provides the most comprehensive view of lincRNAs in Norway spruce and is the first study to perform global identification of lincRNAs during somatic embryogenesis in conifers. The data have been integrated into the expression visualisation tools at the PlantGenIE.org web resource to enable easy access to the community. This will facilitate the use of the data to address novel questions about the role of lincRNAs in the regulation of embryogenesis and facilitate future comparative genomics studies.

TRNA-derived fragment 3031B regulates human anterior cruciate ligament cell proliferation and survival by targeting RELA

tRNA-derived fragments (tRFs) are novel short noncoding RNAs that play pivotal roles in cell proliferation and survival. However, knowledge of the biological roles of tRFs in anterior cruciate ligament (ACL) cells is limited. Here, we intended to investigate the function of tRF-3031B in ACL cell. We used the tRF and tiRNA array to analyze tRF and tiRNA expression profiles in osteoarthritis (OA) ACL cells and normal ACL cells, and qRT-PCR and fluorescence in situ hybridization (FISH) were used to determine tRF-3031B expression. The results showed that tRF-3031B was expressed at low levels in OA ACL and Interleukin-1β (IL-1β) treated ACL cells. We found that RELA was the target of tRF-3031B. When ACL cells were transfected with tRF-3031B mimics, RELA expression was suppressed, whereas transfection with tRF-3031B inhibitors had the opposite effect. The rescue and dual-luciferase reporter assays showed that tRF-3031B silenced the RELA expression by binding to its untranslated region (3′-UTR). Hence, this study showed the novel function of tRF-3031B in regulating ACL cell proliferation and survival by targeting RELA, and these findings may offer a new direction for the study of ACL degeneration and pathophysiological of OA.

Understanding the role of miRNAs in cervical cancer pathogenesis and therapeutic responses.

Cervical cancer (CC) is the most common cancer in women and poses a serious threat to health. Despite familiarity with the factors affecting its etiology, initiation, progression, treatment strategies, and even resistance to therapy, it is considered a significant problem for women. However, several factors have greatly affected the previous aspects of CC progression and treatment in recent decades. miRNAs are short non-coding RNA sequences that regulate gene expression by inhibiting translation of the target mRNA. miRNAs play a crucial role in CC pathogenesis by promoting cancer stem cell (CSC) proliferation, postponing apoptosis, continuing the cell cycle, and promoting invasion, angiogenesis, and metastasis. Similarly, miRNAs influence important CC-related molecular pathways, such as the PI3K/AKT/mTOR signaling pathway, Wnt/β-catenin system, JAK/STAT signaling pathway, and MAPK signaling pathway. Moreover, miRNAs affect the response of CC patients to chemotherapy and radiotherapy. Consequently, this review aims to provide an acquainted summary of onco miRNAs and tumor suppressor (TS) miRNAs and their potential role in CC pathogenesis and therapy responses by focusing on the molecular pathways that drive them.

The role of tRF-Val-CAC-010 in lung adenocarcinoma: implications for tumorigenesis and metastasis

ObjectiveTransfer RNA-derived fragments (tRFs) are short non-coding RNA (ncRNA) sequences, ranging from 14 to 30 nucleotides, produced through the precise cleavage of precursor and mature tRNAs. While tRFs have been implicated in various diseases, including cancer, their role in lung adenocarcinoma (LUAD) remains underexplored. This study aims to investigate the impact of tRF-Val-CAC-010, a specific tRF molecule, on the phenotype of LUAD cells and its role in tumorigenesis and progression in vivo.MethodsThe expression level of tRF-Val-CAC-010 was quantified using quantitative real-time polymerase chain reaction (qRT-PCR). Specific inhibitors and mimics of tRF-Val-CAC-010 were synthesized for transient transfection. Cell proliferation was assessed using the Cell Counting Kit-8 (CCK-8), while cell invasion and migration were evaluated through Transwell invasion and scratch assays. Flow cytometry was utilized to analyze cell cycle and apoptosis. The in vivo effects of tRF-Val-CAC-010 on tumor growth and metastasis were determined through tumor formation and metastasis imaging experiments in nude mice.ResultsThe expression level of tRF-Val-CAC-010 was upregulated in A549 and PC9 LUAD cells (P < 0.01). Suppression of tRF-Val-CAC-010 expression resulted in decreased proliferation of A549 and PC9 cells (P < 0.001), reduced invasion and migration of A549 (P < 0.05, P < 0.001) and PC9 cells (P < 0.05, P < 0.01), enhanced apoptosis in both A549 (P < 0.05) and PC9 cells (P < 0.05), and increased G2 phase cell cycle arrest in A549 cells (P < 0.05). In vivo, the tumor formation volume in the tRF-inhibitor group was significantly smaller than that in the model and tRF-NC groups (P < 0.05). The metastatic tumor flux value in the tRF-inhibitor group was also significantly lower than that in the model and tRF-NC groups (P < 0.05).ConclusionThis study demonstrates that tRF-Val-CAC-010 promotes proliferation, migration, and invasion of LUAD cells and induces apoptosis in vitro, however, its specific effects on the cell cycle require further elucidation. Additionally, tRF-Val-CAC-010 enhances tumor formation and metastasis in vivo. Therefore, tRF-Val-CAC-010 may serve as a novel diagnostic biomarker and potential therapeutic target for LUAD.

RNA-directed peptide synthesis across a nicked loop.

Ribosomal translation at the origin of life requires controlled aminoacylation to produce mono-aminoacyl esters of tRNAs. Herein, we show that transient annealing of short RNA oligo:amino acid mixed anhydrides to an acceptor strand enables the sequential transfer of aminoacyl residues to the diol of an overhang, first forming aminoacyl esters then peptidyl esters. Using N-protected aminoacyl esters prevents unwanted peptidyl ester formation in this manner. However, N-acyl-aminoacyl transfer is not stereoselective.

Synthesis and Properties of RNA Modified with Thioamide Internucleoside Linkage.

Recent success of RNA therapeutics has reinvigorated interest in chemical modifications of RNA. As exemplified by the phosphorothioates, modifications of sugar-phosphate backbone have been remarkably impactful but relatively underexplored in therapeutic RNAs. The present study reports synthesis, thermal stability, and RNA interference activity of RNAs modified with thioamide linkages. Compared to the previously studied amide-modified RNA, thioamide linkages strongly destabilized a short self-complementary RNA model duplex. However, in short interfering RNAs amides and thioamides had a similar effect on duplex stability and target RNA cleavage activity and specificity. Hence, the thioamide may be added to the toolbox of chemical biologist as a useful backbone modification well tolerated by the RNA interference machinery.

Two RNA Folds from One Sequence: A Ribozyme with Versatile Substrate Processing Abilities.

We report the design of a single RNA sequence capable of adopting one of two ribozyme folds and catalyzing the cleavage and/or ligation of the respective substrates. The RNA is able to change its conformation in response to its environment, hence it is called chameleon ribozyme (CHR). Efficient RNA cleavage of two different substrates as well as RNA ligation by CHR is demonstrated in separate experiments and in a one pot reaction. Our study shows that sequence variants of the hairpin ribozyme intersect with the hammerhead ribozyme and that rather short RNA molecules can have comprehensive conformational flexibility, which is an important feature for the emergence of new functional folds in early evolution.

In silico analysis of aptamer-RNA conjugate interactions with human transferrin receptor

The human transmembrane protein Transferrin Receptor-1 is regarded as a promising target for the systemic delivery of therapeutic agents, particularly of nucleic acid therapeutics, such as short double stranded RNAs. This ubiquitous receptor is involved in cellular iron uptake, keeping intracellular homeostasis. It is overexpressed in multiple cancer cell types and is internalized via clathrin-mediated endocytosis. In previous studies, a human transferrin receptor-1 RNA aptamer, identified as TR14 ST1–3, was shown to be capable of effectively internalizing into cells in culture and to deliver small, double stranded RNAs in vitro and in vivo, via systemic administration.To understand, at the molecular level, the aptamer binding to the receptor and the impact of conjugation with the therapeutic RNA, a multi-level in silico protocol was employed, including protein-aptamer docking, molecular dynamics simulations and free energy calculations. The competition for the binding pocket, between the aptamer and the natural ligand human Transferrin, was also evaluated.The results show that the aptamer binds to the same region as Transferrin, with residues from the helical domain showing a critical role. Moreover, the conjugation to the therapeutic RNA, was shown not to affect aptamer binding. Overall, this study provides an atomic-level understanding of aptamer association to human Transferrin Receptor-1 and of its conjugation with a short model-therapeutic RNA, providing also important clues for futures studies aiming to deliver other oligonucleotide-based therapeutics via Transferrin Receptor.