Are Non-Coding RNAs Useful Biomarkers in Parathyroid Tumorigenesis?

Tumors of the parathyroid glands are common endocrine diseases almost always characterized by parathyroid hormone hypersecretion that determines the clinical manifestations of primary hyperparathyroidism, such as fatigue, kidney problems, weakness, brittle bones, and other symptoms. Most parathyroid neoplasia are benign adenomas, although rare malignant forms have been described. They are heterogeneous in terms of clinical presentation and the associated signs and symptoms overlap with those of disease and aging. Furthermore, most patients with hypercalcemia are discovered during routine blood tests for other reasons. Surgical removal is considered the main therapeutic option to cure these endocrine tumors and, therefore, innovative therapeutic approaches are actively required. Recently, a growing number of studies have suggested that alterations to the epigenetic mechanisms could play a pivotal role in parathyroid tumorigenesis. Most of the attention has been focused on non-coding RNAs (ncRNAs) (i.e., miRNAs, lncRNAs, and circRNAs) whose expression profile has been found to be deregulated in parathyroid tumors. The aim of the present paper is to give an insight into the ncRNAs involved in parathyroid tumorigenesis, which could be used in the future either as innovative diagnostic biomarkers or as therapeutic targets for the treatment of this endocrine neoplasia.

The Translational Potential of Non-coding RNAs and Multimodal MRI Data Sets as Diagnostic and Differential Diagnostic Biomarkers for Mood Disorders.

Major Depressive Disorder (MDD) and Bipolar Disorder (BD) have a high prevalence and detrimental socio-economic consequences for the patients and the community. Furthermore, the depressive symptomatology of both disorders is essentially identical, thus rendering the clinical differential diagnosis between the two significantly more difficult considering the concomitant lack of objective biomarkers. Mood disorders are multifactorial disorders the pathophysiology of which includes genetic, epigenetic, neurobiological, neuroimmunological, structural and functional brain alterations, etc. Aberrant genetic variants as well as changed differential expression of microRNAs (miRNAs) and long non-coding RNAs (lncRNAs) have been implicated in the pathophysiology of MDD and BD. MiRNAs as well as lncRNAs have regulatory and modulating functions on protein-- coding gene expression thus influencing the remodeling of the architecture, neurotransmission, immunomodulation, etc. in the Central Nervous System (CNS) which are essential in the development of psychiatric disorders including MDD and BD. Moreover, both shared and distinct structural, connectivity, task-related and metabolic features have been observed via functional magnetic resonance imaging and magnetic resonance spectroscopy, suggesting the possibility of a dimensional continuum between the two disorders instead of a categorical differentiation. Aberrant connectivity within and between the Default Mode Network, the Salience Network, Executive Network, etc. as well as dysfunctional emotion, cognitive and executive processing have been associated with mood disorders. Therefore, the aim of this review is to explore a more multidimensional framework in the scientific research of mood disorders, including epigenetic and neuroimaging data in order to shape an outline for their translational capacity in clinical practice.

Methylation status of nc886 epiallele reflects periconceptional conditions and is associated with glucose metabolism through nc886 RNAs.

BACKGROUND: Non-coding RNA 886 (nc886) is coded from a maternally inherited metastable epiallele. We set out to investigate the determinants and dynamics of the methylation pattern at the nc886 epiallele and how this methylation status associates with nc886 RNA expression. Furthermore, we investigated the associations between the nc886 methylation status or the levels of nc886 RNAs and metabolic traits in the YFS and KORA cohorts. The association between nc886 epiallele methylation and RNA expression was also validated in induced pluripotent stem cell (iPSC) lines. RESULTS: We confirm that the methylation status of the nc886 epiallele is mostly binomial, with individuals displaying either a non- or hemi-methylated status, but we also describe intermediately and close to fully methylated individuals. We show that an individual's methylation status is associated with the mother's age and socioeconomic status, but not with the individual's own genetics. Once established, the methylation status of the nc886 epiallele remains stable for at least 25 years. This methylation status is strongly associated with the levels of nc886 non-coding RNAs in serum, blood, and iPSC lines. In addition, nc886 methylation status associates with glucose and insulin levels during adolescence but not with the indicators of glucose metabolism or the incidence of type 2 diabetes in adulthood. However, the nc886-3p RNA levels also associate with glucose metabolism in adulthood. CONCLUSIONS: These results indicate that nc886 metastable epiallele methylation is tuned by the periconceptional conditions and it associates with glucose metabolism through the expression of the ncRNAs coded in the epiallele region.

Assessing the involvement of the placental microbiome and virome in preeclampsia using non coding RNA sequencing.

OBJECTIVES: Preeclampsia is a dangerous pregnancy complication. The source of preeclampsia is unknown, though the placenta is believed to have a central role in its pathogenesis. An association between maternal infection and preeclampsia has been demonstrated, yet the involvement of the placental microbiome in the etiology of preeclampsia has not been determined. In this study, we examined whether preeclampsia is associated with an imbalanced microorganism composition in the placenta. METHODS: To this end, we developed a novel method for the identification of bacteria/viruses based on sequencing of small non-coding RNA, which increases the microorganism-to-host ratio, this being a major challenge in microbiome methods. We validated the method on various infected tissues and demonstrated its efficiency in detecting microorganisms in samples with extremely low bacterial/viral biomass. We then applied the method to placenta specimens from preeclamptic and healthy pregnancies. Since the placenta is a remarkably large and heterogeneous organ, we explored the bacterial and viral RNA at each of 15 distinct locations. RESULTS: Bacterial RNA was detected at all locations and was consistent with previous studies of the placental microbiome, though without significant differences between the preeclampsia and control groups. Nevertheless, the bacterial RNA composition differed significantly between various areas of the placenta. Viral RNA was detected in extremely low quantities, below the threshold of significance, thus viral abundance could not be determined. CONCLUSIONS: Our results suggest that the bacterial and viral abundance in the placenta may have only limited involvement in the pathogenesis of preeclampsia. The evidence of a heterogenic bacterial RNA composition in the various placental locations warrants further investigation to capture the true nature of the placental microbiome.

Comprehensive discovery of novel structured noncoding RNAs in 26 bacterial genomes.

Comparative sequence analysis methods are highly effective for uncovering novel classes of structured noncoding RNAs (ncRNAs) from bacterial genomic DNA sequence datasets. Previously, we developed a computational pipeline to more comprehensively identify structured ncRNA representatives from individual bacterial genomes. This search process exploits the fact that genomic regions serving as templates for the transcription of structured RNAs tend to be present in longer than average noncoding 'intergenic regions' (IGRs) that are enriched in G and C nucleotides compared to the remainder of the genome. In the present study, we apply this computational pipeline to identify structured ncRNA candidates from 26 diverse bacterial species. Numerous novel structured ncRNA motifs were discovered, including several riboswitch candidates, one whose ligand has been identified and others that have yet to be experimentally validated. Our findings support recent predictions that hundreds of novel ribo-switch classes and other ncRNAs remain undiscovered among the limited number of bacterial species whose genomes have been completely sequenced.

Computational Analysis of circRNA Expression Data.

Analysis of circular RNA (circRNA) expression from RNA-Seq data can be performed with different algorithms and analysis pipelines, tools allowing the extraction of heterogeneous information on the expression of this novel class of RNAs. Computational pipelines were developed to facilitate the analysis of circRNA expression by leveraging different public tools in easy-to-use pipelines. This chapter describes the complete workflow for a computationally reproducible analysis of circRNA expression starting for a public RNA-Seq experiment. The main steps of circRNA prediction, annotation, classification, sequence reconstruction, quantification, and differential expression are illustrated.

Exploring Noninvasive Biomarkers with the miRandola Database: A Tool for Translational Medicine.

Noninvasive biomarkers are required for addressing crucial clinical needs. The ideal biomarker should be easily accessible and provide a unique characteristic for a healthy status or a pathological condition. In the last years, microRNAs (miRNAs) have been proposed as promising tissue-based biomarkers for several diseases such as cancer and cardiovascular diseases. Recently, miRNAs have shown great potential as novel noninvasive biomarkers, due to their high stability in human body fluids such as serum, plasma, and urine. Furthermore, many other noncoding RNAs (ncRNAs) such as long noncoding RNAs (lncRNAs) and circular RNAs (circRNAs) have shown to be novel biomarkers as well. The aim of this exciting research field is to offer novel tools, allowing translational scientists to develop new strategies for diagnosis, screening, and monitoring of diseases. In this book chapter, the miRandola database and its applications will be introduced. The database offers the possibility to explore information on ncRNAs as noninvasive biomarkers, manually extracted from scientific literature and public available resources.

Identification and validation of potential mRNA- microRNA- long-noncoding RNA (mRNA-miRNA-lncRNA) prognostic signature for cervical cancer.

Cervical cancer is one of the most common causes of cancer deaths in women due to poor prognosis and high mortality rates. A novel mRNA-miRNA-lncRNA signature linked to prognosis of cervical cancer is needed to help clinicians judge the prognosis of individual patients more accurately. On the basis of GEO datasets, a total of 161 upregulated and 242 downregulated DE-mRNAs were identified firstly. Among them, eight potential biomarkers were found to have prognostic values with cervical cancer and miRNAs-lncRNAs related to these biomarkers were then analyzed to create mRNA-miRNA-lncRNA networks in cervical cancer. Moreover, in vitro experiments such as qRT-PCR, western blot and Edu assays were also performed to validate these promising targets. On the basis of these findings, a total of eight mRNA-miRNA-lncRNA subnetworks were finally established as a novel mRNA-miRNA-lncRNA signature and independent prognostic indicator of clinically relevant parameters by ROC analysis, univariate and multivariate Cox regression. Since some work of validation was done, it is believed that this mRNA-miRNA-lncRNA prognostic signature may be applied as a potential clinical judgment to estimate the prognosis of cervical cancer.

Emerging roles of non­coding RNAs in the response of rectal cancer to radiotherapy (Review).

Radiotherapy (RT) followed by radical surgery is an effective standard treatment strategy for various types of cancer, including rectal cancer. The response to RT varies among patients, and the radiosensitivity of cancer cells determines the clinical outcome of patients. However, the application of RT to patients with radioresistant tumors may result in radiation­induced toxicity without clinical benefits. Currently, there are no effective methods to predict the response to RT. The limitations of the methods currently used to evaluate tumor radiosensitivity, which are mainly based on clinical and radiological features, are low sensitivity and specificity. Non­coding RNAs (ncRNAs) have emerged as a class of biomarkers for predicting radiosensitivity. In particular, the expression pattern of ncRNAs can predict the response to RT in patients with rectal cancer. Thus, ncRNAs may be used as potential biomarkers and therapeutic targets to improve the diagnosis and treatment outcome of patients with rectal cancer. In the present review, the current knowledge on the limitations of RT for rectal cancer and the association between ncRNA expression and sensitivity of rectal cancer to RT are presented. Additionally, the potential of ncRNAs as predictive biomarkers and therapeutic targets to mitigate resistance of rectal cancer to RT is discussed.

Identification and Characterization of Extrachromosomal Circular DNA in Human Placentas With Fetal Growth Restriction.

Many studies have confirmed that extrachromosomal circular DNAs (eccDNAs/ecDNAs) exist in tumor and normal cells independently of the chromosome and are essential for oncogene plasticity and drug resistance. Studies have confirmed that there are many eccDNAs/ecDNAs in maternal plasma derived from the fetus. Fetal growth restriction (FGR) is a pregnancy-related disease associated with high newborn morbidity and mortality. However, the characteristics and nature of eccDNAs/ecDNAs in FGR are poorly understood. This study aims to deconstruct the properties and potential functions of eccDNAs/ecDNAs in FGR. We performed circle-seq to identify the expression profile of eccDNAs/ecDNAs, analyzed by bioinformatics, and verified by real-time Polymerase Chain Reaction (PCR) combined with southern blot in FGR compared with the normal groups. A total of 45,131 eccDNAs/ecDNAs (including 2,118 unique ones) were identified, which had significantly higher abundance in FRG group than in normal group, and was bimodal in length, peaking at ~146bp and ~340bp, respectively. Gestational age may be one independent factor affecting the production of eccDNAs/ecDNAs, most of which come from genomic regions with high gene density, with a 4~12bp repeat around the junction, and their origin had a certain genetic preference. In addition, some of the host-genes overlapped with non-coding RNAs (ncRNAs) partially or even completely. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis revealed that host-genes on the differentially expressed eccDNAs/ecDNAs (DEEECs/DEECs) were mainly enriched in immune-related functions and pathways. The presence of some ecDNAs were verified, and whose variability were consistent with the circle-seq results. We identified and characterized eccDNAs/ecDNAs in placentas with FGR, and elucidated the formation mechanisms and the networks with ncRNAs, which provide a new vision for the screening of new biomarkers and therapeutic targets for FGR.

Levels of Breast Milk MicroRNAs and Other Non-Coding RNAs Are Impacted by Milk Maturity and Maternal Diet.

There is emerging evidence that non-coding RNAs (ncRNAs) within maternal breast milk (MBM) impart unique metabolic and immunologic effects on developing infants. Most studies examining ncRNAs in MBM have focused on microRNAs. It remains unclear whether microRNA levels are related to other ncRNAs, or whether they are impacted by maternal characteristics. This longitudinal cohort study examined 503 MBM samples from 192 mothers to: 1) identify the most abundant ncRNAs in MBM; 2) examine the impact of milk maturity on ncRNAs; and 3) determine whether maternal characteristics affect ncRNAs. MBM was collected at 0, 1, and 4 months post-delivery. High throughput sequencing quantified ncRNAs within the lipid fraction. There were 3069 ncRNAs and 238 microRNAs with consistent MBM presence (≥10 reads in ≥10% samples). Levels of 17 ncRNAs and 11 microRNAs accounted for 80% of the total RNA content. Most abundant microRNAs displayed relationships ([R]>0.2, adj p< 0.05) with abundant ncRNAs. A large proportion of ncRNAs (1269/3069; 41%) and microRNAs (206/238; 86%) were affected by MBM maturity. The majority of microRNAs (111/206; 54%) increased from 0-4 months. Few ncRNAs and microRNAs were affected (adj p < 0.05) by maternal age, race, parity, body mass index, gestational diabetes, or collection time. However, nearly half of abundant microRNAs (4/11) were impacted by diet. To our knowledge this is the largest study of MBM ncRNAs, and the first to demonstrate a relationship between MBM microRNAs and maternal diet. Such knowledge could guide nutritional interventions aimed at optimizing metabolic and immunologic microRNA profiles within MBM.

The emerging role of super enhancer-derived noncoding RNAs in human cancer.

Super enhancers (SEs) are large clusters of adjacent enhancers that drive the expression of genes which regulate cellular identity; SE regions can be enriched with a high density of transcription factors, co-factors, and enhancer-associated epigenetic modifications. Through enhanced activation of their target genes, SEs play an important role in various diseases and conditions, including cancer. Recent studies have shown that SEs not only activate the transcriptional expression of coding genes to directly regulate biological functions, but also drive the transcriptional expression of non-coding RNAs (ncRNAs) to indirectly regulate biological functions. SE-derived ncRNAs play critical roles in tumorigenesis, including malignant proliferation, metastasis, drug resistance, and inflammatory response. Moreover, the abnormal expression of SE-derived ncRNAs is closely related to the clinical and pathological characterization of tumors. In this review, we summarize the functions and roles of SE-derived ncRNAs in tumorigenesis and discuss their prospective applications in tumor therapy. A deeper understanding of the potential mechanism underlying the action of SE-derived ncRNAs in tumorigenesis may provide new strategies for the early diagnosis of tumors and targeted therapy.

Cross-sectional study of human coding- and non-coding RNAs in progressive stages of Helicobacter pylori infection.

Helicobacter pylori infects 4.4 billion individuals worldwide and is considered the most important etiologic agent for peptic ulcers and gastric cancer. Individual response to H. pylori infection is complex and depends on complex interactions between host and environmental factors. The pathway towards gastric cancer is a sequence of events known as Correa's model of gastric carcinogenesis, a stepwise inflammatory process from normal mucosa to chronic-active gastritis, atrophy, metaplasia and gastric adenocarcinoma. This study examines gastric clinical specimens representing different steps of the Correa pathway with the aim of identifying the expression profiles of coding- and non-coding RNAs that may have a role in Correa's model of gastric carcinogenesis. We screened for differentially expressed genes in gastric biopsies by employing RNAseq, microarrays and qRT-PCR. Here we provide a detailed description of the experiments, methods and results generated. The datasets may help other scientists and clinicians to find new clues to the pathogenesis of H. pylori and the mechanisms of progression of the infection to more severe gastric diseases. Data is available via ArrayExpress.

Lipotoxic Injury Differentially Regulates Brain Microvascular Gene Expression in Male Mice.

The Western diet (WD) and hyperlipidemia are risk factors for vascular disease, dementia, and cognitive impairment. However, the molecular mechanisms are poorly understood. This pilot study investigated the genomic pathways by which the WD and hyperlipidemia regulate gene expression in brain microvessels. Five-week-old C57BL/6J wild type (WT) control and low-density lipoprotein receptor deficient (LDL-R-/-) male mice were fed the WD for eight weeks. Differential gene expression, gene networks and pathways, transcription factors, and non-protein coding RNAs were evaluated by a genome-wide microarray and bioinformatics analysis of laser-captured hippocampal microvessels. The WD resulted in the differential expression of 1972 genes. Much of the differentially expressed gene (DEG) was attributable to the differential regulation of cell signaling proteins and their transcription factors, approximately 4% was attributable to the differential expression of miRNAs, and 10% was due to other non-protein coding RNAs, primarily long non-coding RNAs (lncRNAs) and small nucleolar RNAs (snoRNAs) not previously described to be modified by the WD. Lipotoxic injury resulted in complex and multilevel molecular regulation of the hippocampal microvasculature involving transcriptional and post-transcriptional regulation and may provide a molecular basis for a better understanding of hyperlipidemia-associated dementia risk.

Role of noncoding RNAs in cholangiocarcinoma (Review).

Cholangiocarcinoma (CCA) is a malignant tumour originating from biliary epithelial cells, and is increasing in incidence. Radical surgery is the main treatment. However, the pathogenesis of CCA is unclear. Noncoding RNAs (ncRNAs) are non­protein­coding RNAs produced by genomic transcription that include microRNAs (miRNAs), circular RNAs (circRNAs) and long noncoding RNAs (lncRNAs). They play important roles in gene expression, epigenetic modification, cell proliferation, differentiation and reproduction. ncRNAs also serve key roles in cancer development. Numerous studies have been carried out on ncRNAs, and associated publications have shown that ncRNAs are closely associated with the physiological and pathological mechanisms of CCA. The findings of these studies can provide new insights into the diagnosis, treatment and prognosis of CCA. The present review summarizes the pathophysiological mechanisms of different types of ncRNAs, including miRNAs, circRNAs and lncRNAs in CCA, and their applications in the diagnosis and treatment of CCA.

The Emerging Role of Noncoding RNAs in Pediatric Inflammatory Bowel Disease.

Prevalence of inflammatory bowel disease (IBD), a chronic inflammatory disorder of the gut, has been on the rise in recent years-not only in the adult population but also especially in pediatric patients. Despite the absence of curative treatments, current therapeutic options are able to achieve long-term remission in a significant proportion of cases. To this end, however, there is a need for biomarkers enabling accurate diagnosis, prognosis, and prediction of response to therapies to facilitate a more individualized approach to pediatric IBD patients. In recent years, evidence has continued to evolve concerning noncoding RNAs (ncRNAs) and their roles as integral factors in key immune-related cellular pathways. Specific deregulation patterns of ncRNAs have been linked to pathogenesis of various diseases, including pediatric IBD. In this article, we provide an overview of current knowledge on ncRNAs, their altered expression profiles in pediatric IBD patients, and how these are emerging as potentially valuable clinical biomarkers as we enter an era of personalized medicine.

Quantitative analysis of noncoding RNA from paired fresh and formalin-fixed paraffin-embedded brain tissues.

Formalin-fixed paraffin-embedded (FFPE) tissues are commonly used both clinically and in forensic pathology. Recently, noncoding RNA (ncRNA) has attracted interest among molecular medical researchers. However, it remains unclear whether newly identified ncRNAs, such as long noncoding RNA (lncRNA) and circular RNA (circRNA), remain stable for downstream molecular analysis in FFPE tissues. Here, we assessed the feasibility of using autoptic FFPE brain tissues from eight individuals to perform quantitative molecular analyses. Selected RNA targets (9 mRNAs and 15 ncRNAs) with different amplicon lengths were studied by RT-qPCR in paired fresh and FFPE specimens. For RNA quality assessment, RNA purity and yield were comparable between the two sample cohorts; however, the RNA integrity number decreased significantly during FFPE sampling. Amplification efficiency also displayed certain variability related with amplicon length and RNA species. We found molecular evidence that short amplicons of mRNA, lncRNA, and circRNA were amplified more efficiently than long amplicons. With the assistance of RefFinder, 5S, SNORD48, miR-103a, and miR-125b were selected as reference genes given their high stability. After normalization, we found that short amplicon markers (e.g., ACTB mRNA and MALAT1 lncRNA) exhibited high consistency of quantification in paired fresh/FFPE samples. In particular, circRNAs (XPO1, HIPK3, and TMEM56) presented relatively consistent and stable expression profiles in FFPE tissues compared with their corresponding linear transcripts. Additionally, we evaluated the influence of prolonged storage time on the amplification of gene transcripts and found that short amplicons still work effectively in archived FFPE biospecimens. In conclusion, our findings demonstrate the possibility of performing accurate quantitative analysis of ncRNAs using short amplicons and standardized RT-qPCR assays in autopsy-derived FFPE samples.

Fluorescent Visualization of Mango-tagged RNA in Polyacrylamide Gels via a Poststaining Method.

Native and denaturing polyacrylamide gels are routinely used to characterize ribonucleoprotein (RNP) complex mobility and to measure RNA size, respectively. As many gel-imaging techniques use nonspecific stains or expensive fluorophore probes, sensitive, discriminating, and economical gel-imaging methodologies are highly desirable. RNA Mango core sequences are small (19-22 nt) sequence motifs that, when closed by an arbitrary RNA stem, can be simply and inexpensively appended to an RNA of interest. These Mango tags bind with high affinity and specificity to a thiazole-orange fluorophore ligand called TO1-Biotin, which becomes thousands of times more fluorescent upon binding. Here we show that Mango I, II, III, and IV can be used to specifically image RNA in gels with high sensitivity. As little as 62.5 fmol of RNA in native gels and 125 fmol of RNA in denaturing gels can be detected by soaking gels in an imaging buffer containing potassium and 20 nM TO1-Biotin for 30 min. We demonstrate the specificity of the Mango-tagged system by imaging a Mango-tagged 6S bacterial RNA in the context of a complex mixture of total bacterial RNA.

Ro60 and Y RNAs: structure, functions, and roles in autoimmunity.

Ro60, also known as SS-A or TROVE2, is an evolutionarily conserved RNA-binding protein that is found in most animal cells, approximately 5% of sequenced prokaryotic genomes and some archaea. Ro60 is present in cells as both a free protein and as a component of a ribonucleoprotein complex, where its best-known partners are members of a class of noncoding RNAs called Y RNAs. Structural and biochemical analyses have revealed that Ro60 is a ring-shaped protein that binds Y RNAs on its outer surface. In addition to Y RNAs, Ro60 binds misfolded and aberrant noncoding RNAs in some animal cell nuclei. Although the fate of these defective Ro60-bound noncoding RNAs in animal cells is not well-defined, a bacterial Ro60 ortholog functions with 3' to 5' exoribonucleases to assist structured RNA degradation. Studies of Y RNAs have revealed that these RNAs regulate the subcellular localization of Ro60, tether Ro60 to effector proteins and regulate the access of other RNAs to its central cavity. As both mammalian cells and bacteria lacking Ro60 are sensitized to ultraviolet irradiation, Ro60 function may be important during exposure to some environmental stressors. Here we summarize the current knowledge regarding the functions of Ro60 and Y RNAs in animal cells and bacteria. Because the Ro60 RNP is a clinically important target of autoantibodies in patients with rheumatic diseases such as Sjogren's syndrome, systemic lupus erythematosus, and neonatal lupus, we also discuss potential roles for Ro60 RNPs in the initiation and pathogenesis of systemic autoimmune rheumatic disease.

Evolving methods for rational de novo design of functional RNA molecules.

Artificial RNA molecules with novel functionality have many applications in synthetic biology, pharmacy and white biotechnology. The de novo design of such devices using computational methods and prediction tools is a resource-efficient alternative to experimental screening and selection pipelines. In this review, we describe methods common to many such computational approaches, thoroughly dissect these methods and highlight open questions for the individual steps. Initially, it is essential to investigate the biological target system, the regulatory mechanism that will be exploited, as well as the desired components in order to define design objectives. Subsequent computational design is needed to combine the selected components and to obtain novel functionality. This process can usually be split into constrained sequence sampling, the formulation of an optimization problem and an in silico analysis to narrow down the number of candidates with respect to secondary goals. Finally, experimental analysis is important to check whether the defined design objectives are indeed met in the target environment and detailed characterization experiments should be performed to improve the mechanistic models and detect missing design requirements.