Single-stranded Loop Research Articles

Single-Stranded Hairpin Loop RNAs (loopmeRNAs) Potently Induce Gene Silencing through the RNA Interference Pathway.

Synthetic small interfering RNAs conjugated to trivalent N-acetylgalactosamine (GalNAc) are clinically validated drugs for treatment of liver diseases. Incorporation of phosphorothioate linkages and ribose modifications are necessary for stability, potency, and duration of pharmacology. Although multiple alternative siRNA designs such as Dicer-substrate RNA, shRNA, and circular RNA have been evaluated in vitro and in preclinical studies with some success, clinical applications of these designs are limited as it is difficult to incorporate chemical modifications in these designs. An alternative siRNA design that can incorporate chemical modifications through straightforward synthesis without compromising potency will significantly advance the field. Here, we report a facile synthesis of GalNAc ligand-containing single-stranded loop hairpin RNAs (loopmeRNAs) with clinically relevant chemical modifications. We evaluated the efficiency of novel loopmeRNA designs in vivo and correlated their structure-activity relationship with the support of in vitro metabolism data. Sequences and chemical modifications in the loop region of the loopmeRNA design were optimized for maximal potency. Our studies demonstrate that loopmeRNAs can efficiently silence expression of target genes with comparable efficacy to conventional double-stranded siRNAs but reduced environmental and regulatory burdens.

CircSEC24B activates autophagy and induces chemoresistance of colorectal cancer via OTUB1-mediated deubiquitination of SRPX2

Circular RNAs (circRNAs) are a type of regulatory RNA that feature covalently closed single-stranded loops. Evidence suggested that circRNAs play important roles in the progression and development of various cancers. However, the impact of circRNA on autophagy-mediated progression of colorectal cancer (CRC) remains unclear. The objective of this project was to investigate the influence of circSEC24B on autophagy and its underlying mechanisms in CRC. To validate the presence and circular structure of circSEC24B in CRC cells and tissues, PCR and Sanger sequencing techniques were employed. Drug resistance and invasive phenotype of CRC cells were evaluated using CCK8, transwell, and Edu assays. Gain- and loss-of-function experiments were conducted to assess the effects of circSEC24B and its protein partner on the growth, invasion, and metastasis of CRC cells in vitro and in vivo. Interactions between circSEC24B, OTUB1, and SRPX2 were analyzed through immunofluorescence, RNA-pulldown, and RIP assays. Mass spectrometry analysis was used to identify potential binding proteins of circRNA in CRC cells. Vectors were constructed to investigate the specific structural domain of the deubiquitinating enzyme OTUB1 that binds to circSEC24B. Results showed that circSEC24B expression was increased in CRC tissues and cell lines, and it enhanced CRC cell proliferation and autophagy levels. Mechanistically, circSEC24B promoted CRC cell proliferation by regulating the protein stability of SRPX2. Specifically, circSEC24B acted as a scaffold, facilitating the binding of OTUB1 to SRPX2 and thereby enhancing its protein stability. Additionally, evidence suggested that OTUB1 regulated SRPX2 expression through an acetylation-dependent mechanism. In conclusion, this study demonstrated that circSEC24B activated autophagy and induced chemoresistance in CRC by promoting the deubiquitination of SRPX2, mediated by the deubiquitinating enzyme OTUB1.

Rli51 Attenuates Transcription of the Listeria Pathogenicity Island 1 Gene mpl and Functions as a Trans-Acting sRNA in Intracellular Bacteria.

Listeria pathogenicity island 1 (LIPI-1) is a genetic region containing a cluster of genes essential for virulence of the bacterial pathogen Listeria monocytogenes. Main virulence factors in LIPI-1 include long 5' untranslated regions (5'UTRs), among which is Rli51, a small RNA (sRNA) in the 5'UTR of the Zn-metalloprotease-coding mpl. So far, Rli51 function and molecular mechanisms have remained obscure. Here, we show that Rli51 exhibits a dual mechanism of regulation, functioning as a cis- and as a trans-acting sRNA. Under nutrient-rich conditions, rli51-mpl transcription is prematurely terminated, releasing a short 121-nucleotide-long sRNA. Rli51 is predicted to function as a transcription attenuator that can fold into either a terminator or a thermodynamically more stable antiterminator. We show that the sRNA Rli21/RliI binds to a single-stranded RNA loop in Rli51, which is essential to mediate premature transcription termination, suggesting that sRNA binding could stabilize the terminator fold. During intracellular infection, rli51 transcription is increased, which generates a higher abundance of the short Rli51 sRNA and allows for transcriptional read-through into mpl. Comparative intracellular bacterial transcriptomics in rli51-null mutants and the wild-type reference strain EGD-e suggests that Rli51 upregulates iron-scavenging proteins and downregulates virulence factors from LIPI-1. MS2 affinity purification confirmed that Rli51 binds transcripts of the heme-binding protein Lmo2186 and Lmo0937 in vivo. These results prove that Rli51 functions as a trans-acting sRNA in intracellular bacteria. Our research shows a growth condition-dependent mechanism of regulation for Rli51, preventing unintended mpl transcription in extracellular bacteria and regulating genes important for virulence in intracellular bacteria.

The circMYBL2-Encoded p185 Protein Suppresses Colorectal Cancer Progression by Inhibiting Serine Biosynthesis.

Circular RNAs are a class of covalently closed single-stranded loop RNAs that have been implicated to play a functional role in almost all types of cancers. Previous studies have revealed that circMYBL2 acts as a tumor-promoting circular RNA. In this study, we found that circMYBL2 in colorectal cancer encodes a 185-amino acid protein, p185. Functionally, circMYBL2-encoded p185 suppressed the growth and aggressiveness of colorectal cancer cells in vitro and in vivo. Mechanistically, p185 counteracted ubiquitin C-terminal hydrolase L3 (UCHL3)-mediated deubiquitination of phosphoglycerate dehydrogenase (PHGDH) by competitively binding to the C1 domain of UCHL3, resulting in PHGDH degradation and a subsequent reduction in serine and glycine biosynthesis. These data revealed that the circMYBL2-encoded p185 isoform serves as a tumor suppressor to inhibit the progression of colorectal cancer by reducing serine biosynthesis. Significance: A p185 protein encoded by circMYBL2 functions as a tumor suppressor that inhibits the progression of colorectal cancer by increasing the degradation of PHGDH to reduce serine biosynthesis.

Mutational impact of APOBEC3A and APOBEC3B in a human cell line and comparisons to breast cancer.

A prominent source of mutation in cancer is single-stranded DNA cytosine deamination by cellular APOBEC3 enzymes, which results in signature C-to-T and C-to-G mutations in TCA and TCT motifs. Although multiple enzymes have been implicated, reports conflict and it is unclear which protein(s) are responsible. Here we report the development of a selectable system to quantify genome mutation and demonstrate its utility by comparing the mutagenic activities of three leading candidates-APOBEC3A, APOBEC3B, and APOBEC3H. The human cell line, HAP1, is engineered to express the thymidine kinase (TK) gene of HSV-1, which confers sensitivity to ganciclovir. Expression of APOBEC3A and APOBEC3B, but not catalytic mutant controls or APOBEC3H, triggers increased frequencies of TK mutation and similar TC-biased cytosine mutation profiles in the selectable TK reporter gene. Whole genome sequences from independent clones enabled an analysis of thousands of single base substitution mutations and extraction of local sequence preferences with APOBEC3A preferring YTCW motifs 70% of the time and APOBEC3B 50% of the time (Y = C/T; W = A/T). Signature comparisons with breast tumor whole genome sequences indicate that most malignancies manifest intermediate percentages of APOBEC3 signature mutations in YTCW motifs, mostly between 50 and 70%, suggesting that both enzymes contribute in a combinatorial manner to the overall mutation landscape. Although the vast majority of APOBEC3A- and APOBEC3B-induced single base substitution mutations occur outside of predicted chromosomal DNA hairpin structures, whole genome sequence analyses and supporting biochemical studies also indicate that both enzymes are capable of deaminating the single-stranded loop regions of DNA hairpins at elevated rates. These studies combine to help resolve a long-standing etiologic debate on the source of APOBEC3 signature mutations in cancer and indicate that future diagnostic and therapeutic efforts should focus on both APOBEC3A and APOBEC3B.

Basic protein- and peptide-induced stabilization of long-loop DNA G-guadruplexes

The human genome contains many G-quadruplex-forming sequences, including sequences containing long single-stranded loops that are believed to be unfavorable for G-quadruplex formation. The intracellular environment of biological cells is crowded with proteins with charged surfaces. Understanding the effects of protein-rich environments is important for understanding the formation of G-quadruplexes in an intracellular environment. In this study, we investigated the structural stability of DNA G-quadruplexes in the presence of several types of globular proteins (lysozyme, cytochrome c, bovine serum albumin, myoglobin, histone proteins, and serum proteins), unstructured polypeptides (protamine and poly-l-lysine), and oligopeptides (RGG/RG-domain peptides and short repeated peptides). Thermal melting studies of G-quadruplex-forming oligonucleotides derived from the human telomeric repeat sequence revealed that environments containing high concentrations of proteins and peptides differently affected the G-quadruplex stability according to their loop lengths. We found that weak electrostatic interactions of G-quadruplex loops with basic proteins and peptides improved the stability of long-loop G-quadruplexes and the interactions were strengthened under crowded conditions simulated by dextran. The comparison of the effects of different types of proteins and peptides indicated that excluded volume interactions and structural flexibility of both DNA and polypeptide chains influenced the efficiency of their interactions. This study provides insights into long-loop G-quadruplex stability in a crowded intracellular environment and the recognition of G-quadruplexes by arginine-rich domains of G-quadruplex-binding proteins.

Multiple non-canonical base-stacking interactions as one of the major determinants of RNA tertiary structure organization

Interplane (stacking) interactions of heterocyclic bases of nucleotide residues (n.t.) of RNA are one of the most important factors in the organization of its secondary and tertiary structure. Most of these (canonical) interactions are carried out between neighbors in the polynucleotide chains of RNA. However, with the accumulation of data on the atomic tertiary structures of a wide variety of RNAs and their complexes with proteins, it became clear that RNA nucleotide residues that are not neighbors in their polynucleotide chains and are sometimes separated in the RNA primary structure by tens or hundreds of n.t. can interact with the help of base stacking (non-canonical). This paper presents an exhaustive database of such elements and their environment in the macromolecules of natural and synthetic RNAs. They were called nonadjacent base-stacking elements (NA-BSE). The analysis of these data showed that the NA-BSE forming nucleotides, on average, account for about a quarter of all nucleotides of a particular RNA, therefore, they should be considered as real motifs in their tertiary structure. The classification of NA-BSE by types of localization in RNA macromolecules is carried out. It is shown that the structure-forming role of NA-BSE consists in the compact folding of single-stranded RNA loops, in the transformation of double-stranded bulges into imperfect helices, as well as in the binding of RNA regions removed in their primary and secondary structure.

Multiple Non-Canonical Base-Stacking Interactions as One of the Major Determinants of RNA Tertiary Structure Organization.

Stacking interactions of heterocyclic bases of ribonucleotides are one of the most important factors in the organization of RNA secondary and tertiary structure. Most of these (canonical) interactions are formed between adjacent residues in RNA polynucleotide chains. However, with the accumulation of data on the atomic tertiary structures of various RNAs and their complexes with proteins, it has become clear that nucleotide residues that are not adjacent in the polynucleotide chains and are sometimes separated in the RNA primary structure by tens or hundreds of nucleotides can interact via (non-canonical) base stacking. This paper presents an exhaustive database of such nonadjacent base-stacking elements (NA-BSEs) and their environment in the macromolecules of natural and synthetic RNAs. Analysis of these data showed that NA-BSE-forming nucleotides, on average, account for about a quarter of all nucleotides in a particular RNA and, therefore, should be considered as bona fide motifs of the RNA tertiary structure. We also classified NA-BSEs by their location in RNA macromolecules. It was shown that the structure-forming role of NA-BSEs involves compact folding of single-stranded RNA loops, transformation of double-stranded bulges into imperfect helices, and binding of RNA regions distant in the primary and secondary RNA structure.

Single-molecule assay for highly sensitive SARS-CoV-2 gene detection based on CRISPR-Cas12a.

A trans-cleavage activity of some CRISPR-associated (Cas) proteins including Cas12a and Cas 13a that non-specifically cleaves single-stranded DNA or RNA, has been wildly used to develop sensitive gene detection methods because it is a multiple-turnover enzymatic process, only activated via guide RNA-target gene pairing. Combined with a pre-amplification step, CRISPR-Cas12a-based gene detection methods achieved even attomolar detection limits. However, the pre-amplification step often accompanies the tedious design of primers and inevitable base substitution errors by DNA polymerases, which is not suitable for quantitative analysis. Here, we report a single-molecule assay for ultrasensitive and quantitative gene detection utilizing the trans-cleavage activity of Cas12a without pre-amplification. To monitor the trans-cleavage of Cas12a at the single-molecule level, we designed a hairpin DNA probe with a stem-loop structure where the single-stranded DNA loop can be cleaved via the trans-cleavage of Cas12a, labeled fluorophore and quencher pairs at the end of the stem and immobilized it at the surface. Only in the existence of target gene, the trans-cleavage of Cas12a is activated, cleaving the loop of the hairpin DNA probe, making the quencher leave from the surface, and resulting in the appearance of a fluorescent spot. In other words, the number of appearing fluorescent spots reflects the amount of target gene quantitatively. After optimization of various experimental conditions such as different Cas12a orthologs, monovalent and divalent ion concentration in the reaction buffer, reaction temperature and the mixing ratio of Cas12a to guide RNA, we successfully achieved to detect the E gene of SARS-CoV-2 down to sub picomolar range without any pre-amplification step.

SOD2 in platelets: with age comes responsibility

SOD2 in platelets: with age comes responsibility

Non-Covalent Linkage of Helper Functions to Dumbbell-Shaped DNA Vectors for Targeted Delivery.

Covalently closed dumbbell-shaped DNA delivery vectors comprising the double-stranded gene(s) of interest and single-stranded hairpin loops on both ends represent a safe, stable and efficacious alternative to viral and other non-viral DNA-based vector systems. As opposed to plasmids and DNA minicircles, dumbbells can be conjugated via the loops with helper functions for targeted delivery or imaging. Here, we investigated the non-covalent linkage of tri-antennary N-acetylgalactosamine (GalNAc3) or a homodimer of a CD137/4-1BB-binding aptamer (aptCD137-2) to extended dumbbell vector loops via complementary oligonucleotides for targeted delivery into hepatocytes or nasopharyngeal cancer cells. Enlarging the dumbbell loop size from 4 to 71 nucleotides for conjugation did not impair gene expression. GalNAc3 and aptCD137-2 residues were successfully attached to the extended dumbbell loop via complementary oligonucleotides. DNA and RNA oligonucleotide-based dumbbell-GalNAc3 conjugates were taken up from the cell culture medium by hepatoblastoma-derived human tissue culture cells (HepG2) with comparable efficiency. RNA oligonucleotide-linked conjugates triggered slightly higher levels of gene expression, presumably due to the RNaseH-mediated linker cleavage, the release of the dumbbell from the GalNAc3 residue and more efficient nuclear targeting of the unconjugated dumbbell DNA. The RNaseH-triggered RNA linker cleavage was confirmed in vitro. Finally, we featured dumbbell vectors expressing liver cancer cell-specific RNA trans-splicing-based suicide RNAs with GalNAc3 residues. Dumbbells conjugated with two GalNAc3 residues triggered significant levels of cell death when added to the cell culture medium. Dumbbell vector conjugates can be explored for targeted delivery and gene therapeutic applications.

A Fluorescent Biosensor for Streptavidin Detection Based on Double-Hairpin DNA-Templated Copper Nanoparticles

In this paper, we developed a sensitive, label-free and facile fluorescent strategy for detecting streptavidin (SA) based on double-hairpin DNA-templated copper nanoparticles (CuNPs) and terminal protection of small molecule-linked DNA. Herein, a special DNA hairpin probe was designed and synthesized, which contained two poly T single-stranded loops and a nick point in the middle of the stem. Inspired by the concept of the terminal protection interaction, the specific binding of SA to the biotinylated DNA probe can prevent the exonuclease degradation and keep the integrity of DNA probe, which can be used for synthesizing fluorescent CuNPs as a template. Conversely, the DNA probe would be digested by exonucleases and therefore, would fail to form CuNPs without SA. After systematic optimization, the detection range of SA concentration is from 0.5 to 150 nM with a low detection limit of 0.09 nM. Additionally, the proposed method was also successfully applied in the biological samples. Finally, the proposed method is sensitive, effective and simple, and can be potentially applied for predicting diseases and discovering new drugs.

Molecular Beacons - Loop-Mediated Amplification (MB-LAMP).

High specificity has been demonstrated in polymerase chain reaction (PCR) with the use of molecular beacons (MBs) to detect amplified sequences containing mutations or single-nucleotide polymorphisms (SNPs). MBs have been adapted for use with the isothermal nucleic acid amplification technology loop-mediated amplification (LAMP) by targeting single-stranded loop sequences under optimized conditions to enable applications such as plant genotyping. LAMP has several benefits over PCR, such as rapid amplification, single-temperature reaction conditions enabling low-cost equipment, and robustness to certain PCR inhibitors. However, and despite the increased number of primers required, the specificity of LAMP is limited, and false positive results can be problematic. In this chapter, design considerations for molecular beacons in LAMP assays are described, as well as a method for MB-LAMP amplification and detection, with an example of gene sequences in genetically modified (GM) maize samples.

Loop-Mediated Isothermal Amplification: From Theory to Practice.

Increasing the accuracy of pathogen identification and reducing the duration of analysis remain relevant for modern molecular diagnostics up to this day. In laboratory and clinical practice, detection of pathogens mostly relies on methods of nucleic acid amplification, among which the polymerase chain reaction (PCR) is considered the "gold standard." Nevertheless, in some cases, isothermal amplification methods act as an alternative to PCR diagnostics. Upon more than thirty years of the development of isothermal DNA synthesis, the appearance of loop-mediated isothermal amplification (LAMP) has enabled new directions of in-field diagnostics of bacterial and viral infections. This review examines the key characteristics of the LAMP method and corresponding features in practice. We discuss the structure of LAMP amplicons with single-stranded loops, which have the sites for primer annealing under isothermal conditions. The latest achievements in the modification of the LAMP method are analyzed, which allow considering it as a unique platform for creating the next-generation diagnostic assays.

Starting and stopping RNA polymerase III transcription on single-stranded DNA oligonucleotides.

Circularized single-stranded DNA oligonucleotides, or coligos, show promise as promoter-independent RNA polymerase III (Pol III) transcription templates for generating small RNA in human cells. Using a modified small RNA-seq method, we studied the sequence and secondary structure characteristics that determine Pol III initiation and termination on six coligo templates. The coligos each consisted of an imperfectly base-paired stem flanked by one larger and one smaller loop and were unrelated in sequence. Small RNA-seq data from Pol III coligo transcripts revealed a strong preference for initiating transcription within a 5-nucleotide (nt) window spanning the stem-larger loop junction (loop size 11-24 nt). Transcription in all cases proceeded into the stem rather than into the larger loop, indicating the junction is a site-specific, secondary structure-based Pol III transcription initiator. On average, 81% of sequencing reads showed initiation within this 5 nt junction region, with a template start site nucleotide preference of C > T >> A > G, and a requirement for a template purine at Tss-1. Termination was less precise than initiation and occurred in the larger loop at the same end of the stem where transcription initiated. Termination efficiency was on average 82% and was distributed among the first 11 single-stranded larger loop nt following the stem. The size heterogeneity of Pol III coligo transcripts is thus mainly due to 3' end heterogeneity, whereas the RNA 5' ends were more predictable and homogeneous. Transcription termination did not require an oligo dA template sequence, indicating that termination in this context may be mechanistically different than Pol III's normal gene-context termination. A stepwise model for coligo transcription by Pol III is proposed.

Induction of APOBEC3-mediated genomic damage in urothelium implicates BK polyomavirus (BKPyV) as a hit-and-run driver for bladder cancer

Limited understanding of bladder cancer aetiopathology hampers progress in reducing incidence. Mutational signatures show the anti-viral apolipoprotein B mRNA editing enzyme catalytic polypeptide (APOBEC) enzymes are responsible for the preponderance of mutations in bladder tumour genomes, but no causative viral agent has been identified. BK polyomavirus (BKPyV) is a common childhood infection that remains latent in the adult kidney, where reactivation leads to viruria. This study provides missing mechanistic evidence linking reactivated BKPyV-infection to bladder cancer risk. We used a mitotically-quiescent, functionally-differentiated model of normal human urothelium to examine BKPyV-infection. BKPyV-infection led to significantly elevated APOBEC3A and APOBEC3B protein, increased deaminase activity and greater numbers of apurinic/apyrimidinic sites in the host urothelial genome. BKPyV Large T antigen (LT-Ag) stimulated re-entry from G0 into the cell cycle through inhibition of retinoblastoma protein and activation of EZH2, E2F1 and FOXM1, with cells arresting in G2. The single-stranded DNA displacement loops formed in urothelial cells during BKPyV-infection interacted with LT-Ag to provide a substrate for APOBEC3-activity. Addition of interferon gamma (IFNγ) to infected urothelium suppressed expression of the viral genome. These results support reactivated BKPyV infections in adults as a risk factor for bladder cancer in immune-insufficient populations.

Abstract P5-12-01: Apobec mutation signature in breast cancer explained by combinatorial action of apobec3a and apobec3b

Abstract Background: Mutations drive the initiation and progression of cancer. A leading druggable source of mutation in cancer is enzymatic deamination of single-stranded DNA cytosines by cellular APOBEC3 enzymes. Cytosine-to-uracil deamination can result in a variety of different mutational outcomes including DNA breakage and chromosomal aberrations as well as single base substitution mutations. The latter are comprised of C-to-T and C-to-G mutations in TCA or TCT trinucleotides and attributable to the intrinsic preference of several APOBEC3 family members for binding to these motifs. This mutation pattern, commonly called the “APOBEC signature”, is evident in approximately one-quarter of primary breast tumors and one-third of metastatic breast tumors. Although multiple APOBEC3 enzymes have been implicated as a source of this signature in breast cancer (namely, APOBEC3A, APOBEC3B, and APOBEC3H), the literature is full of conflicting views and it is not clear which of these enzymes contributes most significantly to the mutational landscape of breast cancer. Methods: The near-haploid human cell line, HAP1, was engineered to express the HSV-1 TK gene as a mutation reporter. Candidate APOBEC3 enzymes were expressed individually and confirmed by immunoblotting and activity assays. DNA breakage was measured directly by COMET assays and DNA damage responses indirectly by phosphorylated gamma-H2AX staining. Mutation frequencies were quantified by assaying rates of drug resistance, and mutation patterns were analyzed by sequencing locally in TK and globally across whole genomes. Results: APOBEC3A and APOBEC3B both caused significant increases in chromosomal DNA breakage and DNA damage responses. These enzymes also elevated drug resistance mutation frequencies. In contrast, expression of active APOBEC3H or catalytic mutant derivatives of APOBEC3A and APOBEC3B failed to trigger increases beyond normal spontaneous levels. Interestingly, APOBEC3A and APOBEC3B both inflicted mutation signatures that were indistinguishable locally in TK and globally across whole genomes. The vast majority of these APOBEC signature mutations were dispersed (non-kataegic) and not associated with obvious mesoscale chromosomal features such as single-stranded loop regions of stem-loop structures. Computational comparisons of the broader pentanucleotide APOBEC3A and APOBEC3B mutation signatures and those extracted from 794 primary breast tumor genomes (ICGC cohort) revealed an APOBEC3A-biased subset, an APOBEC3B-biased subset, and a larger group of tumors best explained by combinatorial action of both of these enzymes. Conclusions: Our results indicate that APOBEC3A and APOBEC3B contribute combinatorially in most instances to the observed APOBEC mutation signature in breast cancer. These results provide a framework for developing diagnostic and therapeutic approaches for APOBEC-positive breast cancer. Citation Format: Reuben S Harris, Matthew C Jarvis, Michael A Carpenter, Margaret R Brown, Prokopios P Argyris, William Brown, Douglas Yee. Apobec mutation signature in breast cancer explained by combinatorial action of apobec3a and apobec3b [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-12-01.

Prediction of the RNA Tertiary Structure Based on a Random Sampling Strategy and Parallel Mechanism

Background: Macromolecule structure prediction remains a fundamental challenge of bioinformatics. Over the past several decades, the Rosetta framework has provided solutions to diverse challenges in computational biology. However, it is challenging to model RNA tertiary structures effectively when the de novo modeling of RNA involves solving a well-defined small puzzle. Methods: In this study, we introduce a stepwise Monte Carlo parallelization (SMCP) algorithm for RNA tertiary structure prediction. Millions of conformations were randomly searched using the Monte Carlo algorithm and stepwise ansatz hypothesis, and SMCP uses a parallel mechanism for efficient sampling. Moreover, to achieve better prediction accuracy and completeness, we judged and processed the modeling results. Results: A benchmark of nine single-stranded RNA loops drawn from riboswitches establishes the general ability of the algorithm to model RNA with high accuracy and integrity, including six motifs that cannot be solved by knowledge mining–based modeling algorithms. Experimental results show that the modeling accuracy of the SMCP algorithm is up to 0.14 Å, and the modeling integrity on this benchmark is extremely high. Conclusion: SMCP is an ab initio modeling algorithm that substantially outperforms previous algorithms in the Rosetta framework, especially in improving the accuracy and completeness of the model. It is expected that the work will provide new research ideas for macromolecular structure prediction in the future. In addition, this work will provide theoretical basis for the development of the biomedical field.

Immunocapture-Reverse Transcriptase Loop-Mediated Isothermal Amplification Assay for Detection of Plant RNA Viruses.

Loop-mediated isothermal amplification (LAMP) is a sensitive method that can rapidly amplify a specific nucleic acid target with high specificity. The LAMP reaction process has no denaturation step, instead DNA amplification occurs by strand displacement activity of the Bacillus stearothermophilus (Bst) DNA polymerase under isothermal conditions. It utilizes three sets of forward and reverse oligonucleotide primers specific to six distinct sequences on the target gene. These primers are used to generate amplification products that contain single-stranded loops, thereby allowing primers to bind to these sequences without the need for repeated cycles of thermal denaturation. For diagnosis of pathogens with RNA genome, LAMP has been merged with reverse transcription (RT) step to create RT-LAMP. To further reduce the cost of diagnosis and increase the throughput, immunocapture (IC) step was added to develop IC-RT-LAMP assay. Hence, this chapter focuses on utilizing IC-RT-LAMP assay to specifically identify severe strain of a plant virus from field samples.

Thermostability, Tunability, and Tenacity of RNA as Rubbery Anionic Polymeric Materials in Nanotechnology and Nanomedicine-Specific Cancer Targeting with Undetectable Toxicity.

RNA nanotechnology is the bottom-up self-assembly of nanometer-scale architectures, resembling LEGOs, composed mainly of RNA. The ideal building material should be (1) versatile and controllable in shape and stoichiometry, (2) spontaneously self-assemble, and (3) thermodynamically, chemically, and enzymatically stable with a long shelf life. RNA building blocks exhibit each of the above. RNA is a polynucleic acid, making it a polymer, and its negative-charge prevents nonspecific binding to negatively charged cell membranes. The thermostability makes it suitable for logic gates, resistive memory, sensor set-ups, and NEM devices. RNA can be designed and manipulated with a level of simplicity of DNA while displaying versatile structure and enzyme activity of proteins. RNA can fold into single-stranded loops or bulges to serve as mounting dovetails for intermolecular or domain interactions without external linking dowels. RNA nanoparticles display rubber- and amoeba-like properties and are stretchable and shrinkable through multiple repeats, leading to enhanced tumor targeting and fast renal excretion to reduce toxicities. It was predicted in 2014 that RNA would be the third milestone in pharmaceutical drug development. The recent approval of several RNA drugs and COVID-19 mRNA vaccines by FDA suggests that this milestone is being realized. Here, we review the unique properties of RNA nanotechnology, summarize its recent advancements, describe its distinct attributes inside or outside the body and discuss potential applications in nanotechnology, medicine, and material science.