Small Cloning Research Articles

Clinical and Translational Landscape of Viral Gene Therapies.

Gene therapies hold significant promise for treating previously incurable diseases. A number of gene therapies have already been approved for clinical use. Currently, gene therapies are mostly limited to the use of adeno-associated viruses and the herpes virus. Viral vectors, particularly those derived from human viruses, play a critical role in this therapeutic approach due to their ability to efficiently deliver genetic material to target cells. Despite their advantages, such as stable gene expression and efficient transduction, viral vectors face numerous limitations that hinder their broad application. These limitations include small cloning capacities, immune and inflammatory responses, and risks of insertional mutagenesis. This review explores the current landscape of viral vectors used in gene therapy, discussing the different types of DNA- and RNA-based viral vectors, their characteristics, limitations, and current medical and potential clinical applications. The review also highlights strategies to overcome existing challenges, including optimizing vector design, improving safety profiles, and enhancing transgene expression both using molecular techniques and nanotechnologies, as well as by approved drug formulations.

Cloning and Sequencing Eukaryotic Small RNAs.

Small RNAs are ubiquitous regulators of gene expression that participate in nearly all aspects of physiology in a wide range of organisms. There are many different classes of eukaryotic small RNAs that play regulatory roles at every level of gene expression, including transcription, RNA stability, and translation. While eukaryotic small RNAs display diverse functions across and within classes, they are generally grouped functionally based on the machinery required for their biogenesis, the effector proteins they associate with, and their molecular characteristics. The development of techniques to clone and sequence small RNAs has been critical for their identification, yet the ligation-dependent addition of RNA adapters and the use of reverse transcriptase to generate cDNA in traditional library preparation protocols can be unsuitable to detect certain small RNA subtypes. In particular, 3' or 5' chemical modifications that are characteristic of specific types of small RNAs can impede the ligation-dependent addition of RNA adapters, while internal RNA modifications can interfere with accurate reverse transcription. The inability to clone certain small RNA subtypes with traditional protocols results in an inaccurate assessment of small RNA abundance and diversity, where some RNAs appear over-represented and others are not detected. This overview aims to guide users on how to design small RNA cloning workflows in eukaryotes to more accurately capture specific small RNAs of interest. Hence, we discuss the molecular biology underlying the identification and quantitation of small RNAs, explore the limitations of commonly used protocols, and detail the alternative approaches that can be used to enrich specific small RNA classes. © 2022 Wiley Periodicals LLC.

Identification and expression of white spot syndrome virus-encoded microRNAs in infected Penaeus monodon

White spot syndrome virus (WSSV) is the most devastating DNA virus that causes shrimp mortality worldwide. To better understand WSSV and Penaeus monodon interactions, WSSV-encoded microRNAs (wsv-miRs) were identified by small RNA cloning and next-generation sequencing. Ten wsv-miRs were identified and their expressions can be detected in WSSV-infected tissues only. These wsv-miRs and their flanking sequences can spontaneously form hairpin structure as predicted by Mfold. The expression profiles of 6 wsv-miRs increased dependent on time course of WSSV infection and slightly decreased after 48 hours WSSV challenge. The highest expression was wsv-miR-9 followed by miR-13 and -15. Computational prediction of the candidate targets of wsv-miRs revealed possible functions of wsv-miRs in the controlling of apoptosis, transcription and signaling. This finding provided insights into WSSV-host interaction.

PvdN Enzyme Catalyzes a Periplasmic Pyoverdine Modification

Pyoverdines are high affinity siderophores produced by a broad range of pseudomonads to enhance growth under iron deficiency. They are especially relevant for pathogenic and mutualistic strains that inhabit iron-limited environments. Pyoverdines are generated from non-ribosomally synthesized highly modified peptides. They all contain an aromatic chromophore that is formed in the periplasm by intramolecular cyclization steps. Although the cytoplasmic peptide synthesis and side-chain modifications are well characterized, the periplasmic maturation steps are far from understood. Out of five periplasmic enzymes, PvdM, PvdN, PvdO, PvdP, and PvdQ, functions have been attributed only to PvdP and PvdQ. The other three enzymes are also regarded as essential for siderophore biosynthesis. The structure of PvdN has been solved recently, but no function could be assigned. Here we present the first in-frame deletion of the PvdN-encoding gene. Unexpectedly, PvdN turned out to be required for a specific modification of pyoverdine, whereas the overall amount of fluorescent pyoverdines was not altered by the mutation. The mutant strain grew normally under iron-limiting conditions. Mass spectrometry identified the PvdN-dependent modification as a transformation of the N-terminal glutamic acid to a succinamide. We postulate a pathway for this transformation catalyzed by the enzyme PvdN, which is most likely functional in the case of all pyoverdines.

Global Run-On Sequencing (GRO-seq) Library Preparation from Drosophila Ovaries.

In the past decade, deep-sequencing approaches have greatly improved our knowledge of the genome's potential and have become a crucial milestone for new discoveries in genomics. Transcription is the first step of gene expression; therefore, the detection and measurement of transcription rates is of great interest. Here, a detailed protocol for global run-on sequencing (GRO-seq) library preparation from Drosophila ovaries is described. The method relies on rapid isolation of nuclei with halted transcription, then restarting transcription in physiological conditions in the presence of a labeled nucleotide. The newly transcribed nascent RNA is then isolated and cloned using a small RNA cloning protocol. Although it is time-consuming, the global run-on method allows the user to profile the position, orientation and amount of transcriptionally engaged RNA polymerases across the genome, therefore providing a snapshot of genome-wide transcription.

Small non-coding RNA signature in multiple sclerosis patients after treatment with interferon-β.

BackgroundNon-coding small RNA molecules play pivotal roles in cellular and developmental processes by regulating gene expression at the post-transcriptional level. In human diseases, the roles of the non-coding small RNAs in specific degradation or translational suppression of the targeted mRNAs suggest a potential therapeutic approach of post-transcriptional gene silencing that targets the underlying disease etiology. The involvement of non-coding small RNAs in the pathogenesis of neurodegenerative diseases such as Alzheimer’s , Parkinson’s disease and Multiple Sclerosis has been demonstrated. Multiple sclerosis (MS) is an autoimmune disease of the central nervous system, characterized by chronic inflammation, demyelination and scarring as well as a broad spectrum of signs and symptoms. The current standard treatment for SM is interferon ß (IFNß) that is less than ideal due to side effects. In this study we administered the standard IFN-ß treatment to Relapsing-Remitting MS patients, all responder to the therapy; then examined their sncRNA expression profiles in order to identify the ncRNAs that were associated with MS patients’ response to IFNß.Methods40 IFNß treated Relapsing-Remitting MS patients were enrolled. We analyzed the composition of the entire small transcriptome by a small RNA cloning method, using peripheral blood from Relapsing-Remitting MS patients at baseline and 3 and 6 months after the start of IFNß therapy. Real-time qPCR from the same patients group and from 20 additional patients was performed to profile miRNAs expression.ResultsBeside the altered expression of several miRNAs, our analyses revealed the differential expression of small nucleolar RNAs and misc-RNAs.For the first time, we found that the expression level of miR-26a-5p changed related to INF-β response. MiR-26a-5p expression was significantly higher in IFN-β treated RRMS patients at 3 months treatment, keeping quite stable at 6 months treatments.ConclusionsOur results might provide insights into the mechanisms of action of IFN-β treatment in MS and provide fundamentals for the development of new biomarkers and/or therapeutic tools.

Small RNA cloning and sequencing strategy affects host and viral microRNA expression signatures

The establishment of the microRNA (miRNA) expression signatures is the basic element to investigate the role played by these regulatory molecules in the biology of an organism. Marek's disease virus 1 (MDV-1) is an avian herpesvirus that naturally infects chicken and induces T cells lymphomas. During latency, MDV-1, like other herpesviruses, expresses a limited subset of transcripts. These include three miRNA clusters. Several studies identified the expression of virus and host encoded miRNAs from MDV-1 infected cell cultures and chickens. But a high discrepancy was observed when miRNA cloning frequencies obtained from different cloning and sequencing protocols were compared. Thus, we analyzed the effect of small RNA library preparation and sequencing on the miRNA frequencies obtained from the same RNA samples collected during MDV-1 infection of chicken at different steps of the oncoviral pathogenesis. Qualitative and quantitative variations were found in the data, depending on the strategy used. One of the mature miRNA derived from the latency-associated-transcript (LAT), mdv1-miR-M7-5p, showed the highest variation. Its cloning frequency was 50% of the viral miRNA counts when a small scale sequencing approach was used. Its frequency was 100 times less abundant when determined through the deep sequencing approach. Northern blot analysis showed a better correlation with the miRNA frequencies found by the small scale sequencing approach. By analyzing the cellular miRNA repertoire, we also found a gap between the two sequencing approaches. Collectively, our study indicates that next-generation sequencing data considered alone are limited for assessing the absolute copy number of transcripts. Thus, the quantification of small RNA should be addressed by compiling data obtained by using different techniques such as microarrays, qRT-PCR and NB analysis in support of high throughput sequencing data. These observations should be considered when miRNA variations are studied prior addressing functional studies.

Small RNA library cloning procedure for deep sequencing of specific endogenous siRNA classes in Caenorhabditis elegans.

In recent years, distinct classes of small RNAs ranging in size from ~21 to 26 nucleotides have been discovered and shown to play important roles in a wide array of cellular functions. Because of the abundance of these small RNAs, library preparation from an RNA sample followed by deep sequencing provides the identity and quantity of a particular class of small RNAs. In this chapter we describe a detailed protocol for preparing small RNA libraries for deep sequencing on the Illumina platform from the nematode C. elegans.

High-efficiency RNA cloning enables accurate quantification of miRNA expression by deep sequencing

Small RNA cloning and sequencing is uniquely positioned as a genome-wide approach to quantify miRNAs with single-nucleotide resolution. However, significant biases introduced by RNA ligation in current protocols lead to inaccurate miRNA quantification by 1000-fold. Here we report an RNA cloning method that achieves over 95% efficiency for both 5′ and 3′ ligations. It achieves accurate quantification of synthetic miRNAs with less than two-fold deviation from the anticipated value and over a dynamic range of four orders of magnitude. Taken together, this high-efficiency RNA cloning method permits accurate genome-wide miRNA profiling from total RNAs.

MicroRNAs in farm animals

MicroRNAs (miRNAs) are a class of ∼22 nucleotide-long small noncoding RNAs that target mRNAs for translational repression or degradation. miRNAs target mRNAs by base-pairing with the 3′-untranslated regions (3′-UTRs) of mRNAs. miRNAs are present in various species, from animals to plants. In this review, we summarize the identification, expression, and function of miRNAs in four important farm animal species: cattle, chicken, pig and sheep. In each of these species, hundreds of miRNAs have been identified through homology search, small RNA cloning and next generation sequencing. Real-time RT-PCR and microarray experiments reveal that many miRNAs are expressed in a tissue-specific or spatiotemporal-specific manner in farm animals. Limited functional studies suggest that miRNAs have important roles in muscle development and hypertrophy, adipose tissue growth, oocyte maturation and early embryonic development in farm animals. Increasing evidence suggests that single-nucleotide polymorphisms in miRNA target sites or miRNA gene promoters may contribute to variation in production or health traits in farm animals.

Insect MicroRNAs: Biogenesis, expression profiling and biological functions

MicroRNAs (miRNA) are a class of endogenous regulatory RNA molecules 21–24 nucleotides in length that modulate gene expression at the post-transcriptional level via base pairing to target sites within messenger RNAs (mRNA). Typically, the miRNA “seed sequence” (nucleotides 2–8 at the 5′ end) binds complementary seed match sites within the 3′ untranslated region of mRNAs, resulting in either translational inhibition or mRNA degradation. MicroRNAs were first discovered in Caenorhabditis elegans and were shown to be involved in the timed regulation of developmental events. Since their discovery in the 1990s, thousands of potential miRNAs have since been identified in various organisms through small RNA cloning methods and/or computational prediction, and have been shown to play functionally important roles of gene regulation in invertebrates, vertebrates, plants, fungi and viruses. Numerous functions of miRNAs identified in Drosophila melanogaster have demonstrated a great significance of these regulatory molecules. However, elucidation of miRNA roles in non-drosophilid insects presents a challenging and important task.

Preparation of Small RNA Libraries for High-Throughput Sequencing

This protocol details the process of small RNA cloning for sequencing on the Illumina/Solexa sequencing platform, but it can be easily modified for use on other next-generation platforms (e.g., SOLiD, 454). This procedure is designed to clone canonical small RNA molecules with 5'-monophosphate and 3'-hydroxyl termini. Modifications, such as the presence of a 2'-O-methyl group, can reduce efficiency, although not sufficiently to negate the utility of the approach. Other termini modifications, such as a 5' triphosphate or a 3' phosphate, can be altered by enzymatic treatment before cloning.

MicroRNA expression signature in skeletal muscle of Nile tilapia

Nile tilapia, Oreochromis niloticus, is one of the most important cultivated species in freshwater aquaculture due to its fast growth rate. microRNAs (miRNAs) are approximately 22-nt noncoding RNAs that have emerged as key regulators of skeletal muscle development, but the knowledge of the identity of myogenic miRNAs and their targets in tilapia remains limited. Here, we identify 25 conserved miRNAs in tilapia skeletal muscle using small RNA cloning. Among these miRNAs, miR-1, miR-27a, miR-133a, and miR-206 are differentially expressed in different development stages. Antagomir administration results in a profound decrease in endogenous expression of miR-1, miR-133a, miR-27a or miR-206, while the expression of putative target genes, including HDAC4, SRF, Pax3 or Pax7, is found to be significantly increased. The inverse expression correlation between miRNAs and putative target genes provides evidence that miR-1, miR-133a, miR-27a or miR-206 can directly regulate HDAC4, SRF, Pax 3 or Pax 7 in vivo. The finding would shed new light on the molecular mechanism of skeletal muscle development.

Reducing ligation bias of small RNAs in libraries for next generation sequencing.

BackgroundThe use of nucleic acid-modifying enzymes has driven the rapid advancement in molecular biology. Understanding their function is important for modifying or improving their activity. However, functional analysis usually relies upon low-throughput experiments. Here we present a method for functional analysis of nucleic acid-modifying enzymes using next generation sequencing.FindingsWe demonstrate that sequencing data of libraries generated by RNA ligases can reveal novel secondary structure preferences of these enzymes, which are used in small RNA cloning and library preparation for NGS. Using this knowledge we demonstrate that the cloning bias in small RNA libraries is RNA ligase-dependent. We developed a high definition (HD) protocol that reduces the RNA ligase-dependent cloning bias. The HD protocol doubled read coverage, is quantitative and found previously unidentified microRNAs. In addition, we show that microRNAs in miRBase are those preferred by the adapters of the main sequencing platform.ConclusionsSequencing bias of small RNAs partially influenced which microRNAs have been studied in depth; therefore most previous small RNA profiling experiments should be re-evaluated. New microRNAs are likely to be found, which were selected against by existing adapters. Preference of currently used adapters towards known microRNAs suggests that the annotation of all existing small RNAs, including miRNAs, siRNAs and piRNAs, has been biased.

Tigecycline challenge triggers sRNA production in Salmonella enterica serovar Typhimurium

BackgroundBacteria employ complex transcriptional networks involving multiple genes in response to stress, which is not limited to gene and protein networks but now includes small RNAs (sRNAs). These regulatory RNA molecules are increasingly shown to be able to initiate regulatory cascades and modulate the expression of multiple genes that are involved in or required for survival under environmental challenge. Despite mounting evidence for the importance of sRNAs in stress response, their role upon antibiotic exposure remains unknown. In this study, we sought to determine firstly, whether differential expression of sRNAs occurs upon antibiotic exposure and secondly, whether these sRNAs could be attributed to microbial tolerance to antibiotics.ResultsA small scale sRNA cloning strategy of Salmonella enterica serovar Typhimurium SL1344 challenged with half the minimal inhibitory concentration of tigecycline identified four sRNAs (sYJ5, sYJ20, sYJ75 and sYJ118) which were reproducibly upregulated in the presence of either tigecycline or tetracycline. The coding sequences of the four sRNAs were found to be conserved across a number of species. Genome analysis found that sYJ5 and sYJ118 mapped between the 16S and 23S rRNA encoding genes. sYJ20 (also known as SroA) is encoded upstream of the tbpAyabKyabJ operon and is classed as a riboswitch, whilst its role in antibiotic stress-response appears independent of its riboswitch function. sYJ75 is encoded between genes that are involved in enterobactin transport and metabolism. Additionally we find that the genetic deletion of sYJ20 rendered a reduced viability phenotype in the presence of tigecycline, which was recovered when complemented. The upregulation of some of these sRNAs were also observed when S. Typhimurium was challenged by ampicillin (sYJ5, 75 and 118); or when Klebsiella pneumoniae was challenged by tigecycline (sYJ20 and 118).ConclusionsSmall RNAs are overexpressed as a result of antibiotic exposure in S. Typhimurium where the same molecules are upregulated in a related species or after exposure to different antibiotics. sYJ20, a riboswitch, appears to possess a trans-regulatory sRNA role in antibiotic tolerance. These findings imply that the sRNA mediated response is a component of the bacterial response to antibiotic challenge.

MICRORNA REGULATES GENE EXPRESSION DURING FRUIT DEVELOPMENT IN PINEAPPLE

MicroRNAs (miRNA) are small RNAs approximately 21-24 nucleotides in size. They function by regulating gene expression of various plant tissues such as leaf, root and embryo through the mechanism of RNAi silencing. Numerous reports have been published of the role of miRNA in regulating fruit development in tomato, a model organism for fruit development and ripening in climacteric fruits. However, there is a dearth of information on its role in non-climateric fruits, such as pineapple. This paper aims to firstly, isolate and characterize miRNAs in pineapples, and secondly, to reveal differentially expressed miRNA profiles between pineapples and tomatoes. MicroRNAs from a mature green pineapple were isolated using an optimized small RNAs concatamerization and cloning method. 150 clones were sequenced resulting in the identification of a total of 1,706 small RNAs. These small RNAs were screened for conserved microRNAs from other plant species using the miRBase online database. The small RNAs were also screened for differentially expressed miRNAs between pineapple and tomato using the Tomato Functional Genomics Database (TFGD). Twelve conserved microRNA families were identified as regulators of fruit development in pineapples. The most abundant was miR157, which had 58 copies out of the total small RNA pool. This was followed by miR396 (6), miR164 (3), miR166 (3), miR319 (3), miR827 (3), miR159 (2), miR171 (2), miR156 (1), miR162 (1), miR395 (1), and miR529 (1). However, the TFGD search revealed that only miR529 and miR827 were absent in tomato. This may be indicative of the differences in the mechanism of regulation by microRNA between climacteric and non-climacteric fruits.

STRU-Cloning: A Fast, Inexpensive and Efficient Cloning Procedure Applicable to Both Small Scale and Structural Genomics Size Cloning

We have developed a Single-Tube Restriction-based Ultrafiltration (STRU) cloning procedure that updates traditional ligation-dependent cloning to challenge the newer, faster and more efficient ligation-free techniques and could make it the method of choice. STRU-cloning employs centrifugal filter units with membrane of suitable cut off to remove small unwanted DNA fragments created during restriction of plasmids or PCR products. Heat inactivation, of restriction enzymes, followed by DNA ligation is then performed on the filtrate. By removing the agarose gel electrophoresis DNA purification step from the traditional protocol, which is time consuming and is known to be the cause of ligation problems, STRU-cloning becomes fast, very efficient, inexpensive and offers the highest degree of cloning flexibility by using restriction sites and can be performed in a single tube. This novel agarose gel-free cloning procedure provides benefits for both small and large scale cloning projects. Unlike traditional cloning it can be easily implemented as a fully automated process at very low costs.

A Rapid Method for Isolation of Low-Molecular-Weight Rna from Arabidopsis Using Low Salt Concentration Buffer

We have developed a rapid extraction method using low salt concentration buffer for the isolation of low-molecular-weight RNA from Arabidopsis tissues. The method was quick and efficient, and the small scale extraction process took no more than 1 hour, while yield and RNA quality were comparable with those of previously reported. The LMW RNA isolated using this method was high quality, abundant in small RNA and free of high molecular weight RNA. This method can be used to extract low-molecular-weight RNA for the purpose of small RNA cloning and detection, and library construction.

Identification of 21 microRNAs in maize and their differential expression under drought stress

MicroRNAs (miRNAs) are a class of small, non-coding regulatory RNAs that regulate gene expression by guiding target mRNA cleavage or translational inhibition. The negative regulation of miRNAs has been found in response to stress in Arabidopsis, rice and other plants. The sequence of the maize genome has been completed very recently; however, only a few maize miRNAs have been identified and deposited in the miRNA registry database, and the details of their regulation of target genes remain to be elucidated. We identified 21 maize miRNAs using bioinformatics computation and small RNA cloning. They are designated as zma-miR161, zma-miR397, zma-miR446, zma-miR479, zma-miR530, zma-miR776, zma-miR782, zma-miR815a, zma-miR818a, zma-miR820, zma-miR828, zma-miR834, zma-miR1, zma-miR2, zma-miR3, zma-miR4, zma-miR5, zma-miR6, zma-miR7, zma-miR8 and zma-miR9. The characterization of these miRNAs reveals a length of 19–24 nt and the typical secondary structure. Using reverse transcriptional polymerase chain reaction (RT-PCR), 13 of these miRNAs were amplified and validated from total small RNA samples of maize. zma-miR1, zma-miR3, zma-miR6, zma-miR479, zma-miR782, zma-miR815a and zma-miR820 were found to be expressed specifically in drought-tolerant inbred line 87-1 under simulated drought stress, whereas the other six were amplified in line 87-1 and in the sensitive inbred line 200B under drought stress and in a well-watered control. The seven differentially expressed miRNAs are suggested to be associated with the regulation of gene expression under drought stress. Using the plant miRNA target finder software miRU and miRU2, 14 of the 21 miRNAs were identified to target genes encoding 21 proteins, most of which are metabolic enzymes. Seven of them are involved in signal transduction, transcription regulation, and bio- or abio-stress responses. The identification and characterization of 21 miRNAs enrich the maize miRNA database and provide valuable information for functional studies of miRNAs. Key words: MicroRNA, maize, bioinformatics, gene cloning, differential expression, drought stress.

MicroRNAs, the epigenetic memory and climatic adaptation in Norway spruce

*Norway spruce expresses a temperature-dependent epigenetic memory from the time of embryo development, which thereafter influences the timing bud phenology. MicroRNAs (miRNAs)are endogenous small RNAs, exerting epigenetic gene regulatory impacts. We have tested for their presence and differential expression. *We prepared concatemerized small RNA libraries from seedlings of two full-sib families, originated from seeds developed in a cold and warm environment. One family expressed distinct epigenetic effects while the other not. We used available plant miRNA query sequences to search for conserved miRNAs and from the sequencing we found novel ones; the miRNAs were monitored using relative real time-PCR. *Sequencing identified 24 novel and four conserved miRNAs. Further screening of the conserved miRNAs confirmed the presence of 16 additional miRNAs. Most of the miRNAs were targeted to unknown genes. The expression of seven conserved and nine novel miRNAs showed significant differences in transcript levels in the full-sib family showing distinct epigenetic difference in bud set, but not in the nonresponding full-sib family. Putative miRNA targets were studied. *Norway spruce contains a set of conserved miRNAs as well as a large proportion of novel nonconserved miRNAs. The differentially expression of specific miRNAs indicate their putative participation in the epigenetic regulation.