Recognition Of Target Sequence Research Articles

Multiplex CRISPR-Cas Genome Editing: Next-Generation Microbial Strain Engineering.

Genome editing is a crucial technology for obtaining desired phenotypes in a variety of species, ranging from microbes to plants, animals, and humans. With the advent of CRISPR-Cas technology, it has become possible to edit the intended sequence by modifying the target recognition sequence in guide RNA (gRNA). By expressing multiple gRNAs simultaneously, it is possible to edit multiple targets at the same time, allowing for the simultaneous introduction of various functions into the cell. This can significantly reduce the time and cost of obtaining engineered microbial strains for specific traits. In this review, we investigate the resolution of multiplex genome editing and its application in engineering microorganisms, including bacteria and yeast. Furthermore, we examine how recent advancements in artificial intelligence technology could assist in microbial genome editing and engineering. Based on these insights, we present our perspectives on the future evolution and potential impact of multiplex genome editing technologies in the agriculture and food industry.

A programmable quantum dot nanosensor guided by three-way junction skeleton-mediated cascade signal amplification for sensitive detection of circRNAs in breast cancer

Circular RNAs (circRNAs) are endogenous covalent circular molecules that can regulate various biological processes in eukaryotes, and their dysregulation is associated with tumorigenesis. Herein, we demonstrate the construction of a programmable quantum dot (QD) nanosensor guided by three-way junction (TWJ) skeleton-mediated cascade signal amplification for sensitive detection of circRNA in clinical breast tissues. The TWJ probe contains a TWJ primer and a TWJ template. The presence of circRNA can specifically hybridize with TWJ probe to form a stable TWJ skeleton, initiating cascade signal amplification to produce abundant linker probes. The resultant linker probes can bind with Cy5-modified signal probes and biotinylated capture probes to yield the sandwich duplexes. Eventually, the immobilization of sandwich duplexes on the 605QD surface yields the 605QD-dsDNA-Cy5 nanoassembly, resulting in effective fluorescence resonance energy transfer (FRET). This nanosensor is free from exogenous reverse transcription primers and complicated probes design, and it exhibits high amplification efficiency, single-base mismatch specificity, and near-zero background signal. It can detect circRNA with attomolar sensitivity, measure endogenous circRNA at the single-cell level, and discriminate circRNA level in breast cancer tissues from adjacent normal tissues. Moreover, this nanosensor can be expanded to detect other circRNAs (e.g., circHIPK3) by easily changing the corresponding target recognition sequences of TWJ probes, with potential applications in biomedical research and molecular diagnosis.

Photoactivatable CRISPR/Cas12a Sensors for Biomarkers Imaging and Point-of-Care Diagnostics.

In recent years, the CRISPR/Cas12a-based sensing strategy has shown significant potential for specific target detection due to its rapid and sensitive characteristics. However, the "always active" biosensors are often insufficient to manipulate nucleic acid sensing with high spatiotemporal control. It remains crucial to develop nucleic acid sensing devices that can be activated at the desired time and space by a remotely applied stimulus. Here, we integrated photoactivation with the CRISPR/Cas12a system for DNA and RNA detection, aiming to provide high spatiotemporal control for nucleic acid sensing. By rationally designing the target recognition sequence, this photoactivation CRISPR/Cas12a system could recognize HPV16 and survivin, respectively. We combined the lateral flow assay strip test with the CRISPR/Cas12a system to realize the visualization of nucleic acid cleavage signals, displaying potential instant test application capabilities. Additionally, we also successfully realized the temporary control of its fluorescent sensing activity for survivin by photoactivation in vivo, allowing rapid detection of target nucleic acids and avoiding the risk of contamination from premature leaks during storage. Our strategy suggests that the CRISPR/Cas12a platform can be triggered by photoactivation to sense various targets, expanding the technical toolbox for precise biological and medical analysis. This study represents a significant advancement in nucleic acid sensing and has potential applications in disease diagnosis and treatment.

Evolution of the Ikaros family transcription factors: From a deuterostome ancestor to humans

Ikaros family proteins (Ikaros, Helios, Aiolos, Eos) are zinc finger transcription factors essential for the development and function of the adaptive immune system. They also control developmental events in neurons and other cell types, suggesting that they possess crucial functions across disparate cell types. These functions are likely shared among the organisms in which these factors exist, and it is thus important to obtain a view of their distribution and conservation across organisms. How this family evolved remains poorly understood. Here we mined protein, mRNA and DNA databases to identify proteins with DNA-binding domains homologous to that of Ikaros. We show that Ikaros-related proteins exist in organisms from all four deuterostome phyla (chordates, echinoderms, hemichordates, xenacoelomorpha), but not in more distant groups. While most non-vertebrates have a single family member, this family grew to six members in the acoel worm Hofstenia miamia, three in jawless and four in jawed vertebrates. Most residues involved in DNA contact from zinc fingers 2 to 4 were identical across the Ikaros family, suggesting conserved mechanisms for target sequence recognition. Further, we identified a novel KRKxxxPxK/R motif that inhibits DNA binding in vitro which was conserved across the deuterostome phyla. We also identified a EψψxxxψM(D/E)QAIxxAIxYLGA(D/E)xL motif conserved among human Ikaros, Aiolos, Helios and subsets of chordate proteins, and motifs that are specific to subsets of vertebrate family members. Some of these motifs are targets of mutations in human patients. Finally we show that the atypical family member Pegasus emerged only in vertebrates, which is consistent with its function in bone. Our data provide a novel evolutionary perspective for Ikaros family proteins and suggest that they have conserved regulatory functions across deuterostomes.

The forkhead DNA-binding domain binds specific G2-rich RNA sequences

Abstract Transcription factors contain a DNA-binding domain ensuring specific recognition of DNA target sequences. The family of forkhead (FOX) transcription factors is composed of dozens of paralogs in mammals. The forkhead domain (FHD) is a segment of about 100 amino acids that binds an A-rich DNA sequence. Using DNA and RNA PCR-SELEX, we show that recombinant FOXL2 proteins, either wild-type or carrying the oncogenic variant C134W, recognize similar DNA-binding sites. This suggests that the oncogenic variant does not alter the intrinsic sequence-specificity of FOXL2. Most importantly, we show that FOXL2 binds G2-rich RNA sequences whereas it virtually fails to bind similar sequences in DNA chemistry. Interestingly, a statistically significant subset of genes responding to the knock-down of FOXL2/Foxl2 harbor such G2-rich sequences and are involved in crucial signaling pathways and cellular processes. In addition, we show that FOXA1, FOXO3a and chimeric FOXL2 proteins containing the FHD of the former are also able to interact with some of the preferred FOXL2-binding sequences. Our results point to an unexpected and novel characteristic of the forkhead domain, the biological relevance of which remains to be explored.

Construction of Genetically Encoded Light-Up RNA Aptamers for Label-free and Ultrasensitive Detection of CircRNAs in Cancer Cells and Tissues.

Circular RNAs (circRNAs) as endogenous non-coding RNAs are characterized by covalently closed circular structures, and they widely exist in mammalian cells. The aberrant expression of circRNAs may result in various diseases. Herein, we demonstrate the construction of genetically encoded light-up RNA aptamers for ultrasensitive and label-free detection of circRNA mitochondrial tRNA translation optimization 1 (circMTO1) in cancer cells and tissues. The light-up RNA aptamers are generated by proximity ligation-activated recombinase polymerase amplification (RPA)-assisted transcription amplification. When circMTO1 is present, it initiates the proximity ligation reaction, activating RPA to produce numerous long double-stranded DNAs containing T7 promoters. Subsequently, the RPA products are identified by T7 RNA polymerase, initiating the transcription amplification reaction to generate abundant Spinach RNA aptamers. Spinach RNA aptamers can bind with DFHBI (3,5-difluoro-4-hydroxybenzylidene imidazolidinone) dye to produce a distinct fluorescence signal with near-zero background. This biosensor exhibits excellent selectivity and high sensitivity with a limit of detection of 2.54 aM. It can accurately monitor cellular circMTO1 at the single-cell level and discriminate the expression of circMTO1 between breast cancer patient tissues and healthy tissues. Notably, this biosensor can be employed to measure other nucleic acids by altering the corresponding target recognition sequences, providing a valuable platform for cancer diagnosis and biomedical study.

New Target Gene Screening Using Shortened and Random sgRNA Libraries in Microbial CRISPR Interference.

CRISPR interference (CRISPRi) screening has been used for identification of target genes related to specific phenotypes using single-molecular guide RNA (sgRNA) libraries. In CRISPRi screening, the sizes of random sgRNA libraries contained with the original target recognition sequences are large (∼1012). Here, we demonstrated that the length of the target recognition sequence (TRS) can be shortened in sgRNAs from the original 20 nucleotides (N20) to 9 nucleotides (N9) that is still sufficient for dCas9 to repress target genes in the xylose operon of Escherichia coli, regardless of binding to a promoter or open reading frame region. Based on the results, we constructed random sgRNA plasmid libraries with 5'-shortened TRS lengths, and identified xylose metabolic target genes by Sanger sequencing of sgRNA plasmids purified from Xyl- phenotypic cells. Next, the random sgRNA libraries were harnessed to screen for target genes to enhance violacein pigment production in synthetic E. coli cells. Seventeen target genes were selected by analyzing the redundancy of the TRS in sgRNA plasmids in dark purple colonies. Among them, seven genes (tyrR, pykF, cra, ptsG, pykA, sdaA, and tnaA) have been known to increase the intracellular l-tryptophan pool, the precursor of a violacein. Seventeen cells with a single deletion of each target gene exhibited a significant increase in violacein production. These results indicate that using shortened random TRS libraries for CRISPRi can be simple and cost-effective for phenotype-based target gene screening.

Mix-and-Detection Assay with Multiple Cyclic Enzymatic Repairing Amplification for Rapid and Ultrasensitive Detection of Long Noncoding RNAs in Breast Tissues.

Long noncoding RNAs (lncRNAs) are valuable biomarkers and therapeutic targets, and they play essential roles in various pathological and biological processes. So far, the reported lncRNA assays usually suffer from unsatisfactory sensitivity and time-consuming procedures. Herein, we develop a mix-and-read assay based on multiple cyclic enzymatic repairing amplification (ERA) for sensitive and rapid detection of mammalian metastasis-associated lung adenocarcinoma transcript 1 (lncRNA MALAT1). In this assay, we design two three-way junction (3WJ) probes including a 3WJ template and a 3WJ primer to specifically recognize lncRNA MALAT1, and the formation of a stable 3WJ structure induces cyclic ERA to generate triggers. The resulting triggers subsequently hybridize with a free 3WJ template and act as primers to initiate new rounds of cyclic ERA, generating abundant triggers. The hybridization of triggers with signal probes forms stable double-stranded DNA duplexes that can be specifically cleaved by apurinic/apyrimidinic endonuclease 1 to produce a high fluorescence signal. This assay can be carried out in a mix-and-read manner within 10 min under an isothermal condition (50 °C), which is the rapidest and simplest method reported so far for the lncRNA MALAT1 assay. This method can sensitively detect lncRNA MALAT1 with a limit of detection of 0.87 aM, and it can accurately measure endogenous lncRNA MALAT1 at the single-cell level. Moreover, this method can distinguish lncRNA MALAT1 expression in breast cancer patient tissues and their corresponding healthy adjacent tissues. Importantly, the extension of this assay to different RNAs detection can be achieved by simply replacing the corresponding target recognition sequences.

A novel CRISPR/Cas14a-based electrochemical biosensor for ultrasensitive detection of Burkholderia pseudomallei with PtPd@PCN-224 nanoenzymes for signal amplification

In this work, PtPd nanoparticles functionalized porphyrin metal-organic framework nanoenzymes (PtPd@PCN-224 nanoenzymes) are exploited as signal amplification tags to fabricate a rapid and ultrasensitive sensitive CRISPR/Cas14a-based electrochemical biosensor for Burkholderia pseudomallei (B. pseudomallei) specific DNA sequences detection. The prepared PtPd@PCN-224 nanoenzymes not only catalyze the reduction peak current of H2O2 to obtain a strong electrochemical signal output, but also provide massive active sites for the assembly of nucleic acids by Zr–O–P bonds. Besides, the designed target-activated CRISPR/Cas14a is able to recognize the target DNA sequences and further trigger the trans-cleavage of ssDNA for signal amplification. Benefiting from the target-activated CRISPR/Cas14a and PtPd@PCN-224 nanoenzymes, the developed electrochemical biosensor for B. pseudomallei DNA detection exhibits high sensitivity with detection of limit down to 12.8 aM and excellent specificity for distinguishing non-targeted bacteria. Moreover, the CRISPR/Cas14a-based electrochemical detection platform can also apply for other pathogenic bacteria diagnostic by well-designing sgRNA for target sequence recognition, possessing high flexibility and versatility in clinical diagnosis.

Biosensor model based on single hairpin structure for highly sensitive detection of multiple targets.

Nowadays, due to the genetic information carried by nucleic acids, they can serve as a biomarker for the early diagnosis of diseases, including tumors and cardiovascular disease, among others, making genetic testing a hotspot of biomedicine. Therefore, we have designed a universal fluorescence biosensor that can detect multiple DNA sequences with good performance. In our designed biosensor, λ exonuclease is used due to its ability to digest double-stranded DNA from the phosphorylated 5'- end and promote the targeted cycle. The exonuclease is introduced into a DNA hairpin containing a target recognition sequence. Hence, with the target, λ exonuclease-assisted targeted recycling can be activated. The hydrolyzed DNA hairpin triggers a strand displacement reaction between the hairpin probe (H1) and F-Q double DNA strand (F-Q), increasing the distance between the fluorescent chain (F) and quenching chain (Q); thus the fluorescence signal is emitted. It is exciting that the detection limit of the biosensor is 300 fM, which is relatively low, and there is an excellent linear relationship between fluorescence intensity and target concentration. Moreover, the biosensor we designed has universal applicability in the detection of other genes, and the range of RSD is 1.28-2.45%. Hence, it has good application prospects and practical value in the early detection of some diseases and the design of fluorescent biosensors.

Switching the Activity of CRISPR/Cas12a Using an Allosteric Inhibitory Aptamer for Biosensing.

The current CRISPR/Cas12a-based diagnostic techniques focus on designing the crRNA or substrate DNA elements to indirectly switch the trans-cleavage activity of Cas12a responsive to target information. Here, we propose the use of an allosteric DNA probe to directly regulate the trans-cleavage activity of Cas12a and present a method for sensing different types of analytes. An allosteric inhibitor probe is rationally designed to couple the target recognition sequence with the inhibitory aptamer of the CRISPR/Cas12a system and enables binding to a specific target to induce the change of conformation, which leads to the loss of its inhibitory function on Cas12a. As a result, the structure-switchable probe can regulate the degree of activity of Cas12a depending on the dose of target. Scalability of our strategy can be achieved by simply replacing the loop domain with different target recognition sequences. The proposed method was validated by detecting adenosine triphosphate and let-7a, giving the detection limits of 490 nM and 26 pM, respectively, and showing an excellent specificity. We believe that this work exploits a viable approach to use the inhibitory aptamer of Cas12a as a regulatory element for biosensing purposes, enriching the arsenal of CRISPR/Cas12a-based methods for molecular diagnostics and spurring further development and application of aptamers of the CRISPR/Cas system.

A Facile, Label-free and Versatile Fluorescence Sensing Nanoplatform Based on Titanium Carbide Nanosheets for the Detection of Various Targets.

The construction of a universal nanoplatform for sensitive detection of multiple targets of interest is of great importance in different research fields. Herein, by ingeniously integrating the target recognition sequences and G-rich sequences into a single-stranded multifunctional DNA probe and adopting Ti3C2 nanosheets as an efficient fluorescence quencher, a simple, low-cost and easy operation fluorescence sensing nanoplatform was proposed. Without an analytical target, the hydrogen bond and metal chelate interaction between the target recognition region of the DNA probe and Ti3C2 nanosheet induce the selective self-assembly of highly fluorescent thioflavin T (ThT)-intercalated DNA probe onto the surface of Ti3C2 nanosheets, resulting in dramatic decrease of fluorescence emitted by ThT-G-quadruplex. In the presence of a target, the target recognition region will selectively bind with the target and the constrained DNA probe is released from the Ti3C2 nanosheets surface, leading to enhanced fluorescence recovery of ThT-G-quadruplex. As a proof of concept, the sensitive and selective detection of p53 gene, Hg2+, and adenosine with the assistance of Ti3C2 nanosheets-based fluorescence sensing nanoplatform were successfully realized. Moreover, it is also applicable for the evaluation the level of these analytical targets in real samples. By simply switching the recognition sequences of DNA probe, the universal sensing strategy could also be applied for detecting many other types of targets. The simple and universal sensing nanoplatform is expected to promote wide applications in environment monitoring and bioanalysis.

Target sequence recognition and gene regulation by Grainyhead/CP2 transcription factors

Target sequence recognition and gene regulation by Grainyhead/CP2 transcription factors

De novo mutations in the BMP signaling pathway in lambdoid craniosynostosis.

Lambdoid craniosynostosis (CS) is a congenital anomaly resulting from premature fusion of the cranial suture between the parietal and occipital bones. Predominantly sporadic, it is the rarest form of CS and its genetic etiology is largely unexplored. Exome sequencing of 25 kindreds, including 18 parent-offspring trios with sporadic lambdoid CS, revealed a marked excess of damaging (predominantly missense) de novo mutations that account for ~ 40% of sporadic cases. These mutations clustered in the BMP signaling cascade (P = 1.6 × 10-7), including mutations in genes encoding BMP receptors (ACVRL1 and ACVR2A), transcription factors (SOX11, FOXO1) and a transcriptional co-repressor (IFRD1), none of which have been implicated in other forms of CS. These missense mutations are at residues critical for substrate or target sequence recognition and many are inferred to cause genetic gain-of-function. Additionally, mutations in transcription factor NFIX were implicated in syndromic craniosynostosis affecting diverse sutures. Single cell RNA sequencing analysis of the mouse lambdoid suture identified enrichment of mutations in osteoblast precursors (P = 1.6 × 10-6), implicating perturbations in the balance between proliferation and differentiation of osteoprogenitor cells in lambdoid CS. The results contribute to the growing knowledge of the genetics of CS, have implications for genetic counseling, and further elucidate the molecular etiology of premature suture fusion.

Recent Advances in Molecular Point of Care Tests

Molecular point of care tests has become promising methods for the global control of infectious diseases in recent years. Different kinds of point of care testing devices have been introduced into the market in the last decade. They are mainly based on miniaturization and integration of sample processing, nucleic acid amplification, and detection systems. These devices must offer a low-cost, sensitive, specific, and practical analysis to be used especially in low-resource settings. Microfluidics has high potential for handling very small volumes of fluids on a single platform. The key design features for molecular point of care tests in resource-limited settings include isothermal nucleic acid amplification methods to eliminate the need for a thermocycler, lyophilized reagents for long-term stability at high temperature and relatively simple test procedures. CRISPR-Cas-based new generation molecular point of care tests have been developed in recent years. In these tests Cas enzymes are used as highly specific target sequence recognition elements. Collateral cleavage activity of these enzymes cleaves both target sequence and labeled RNA in the mixture and a signal is generated. The ongoing Coronavirus 2019 pandemic has shown the importance of rapid diagnostic tests for the prevention of further spread. Although real-time polymerase chain reaction method was used successfully for the rapid diagnosis during the pandemic, additional rapid and practical tests that could be performed outside the laboratories would provide even faster diagnosis and lighten the burden of test load in large central laboratories. Molecular point of care tests are considered to be one of the best candidates to fill the this gap in the near future. The future technology challenges will be the standardization of the methods and further miniaturization of the system components. In this review article, point-of-care tests adapted from nucleic acid amplification methods with a large number of studies in the literature and tests based on the CRISPR-Cas system which constitutes the newest group and which is among the point-of-care molecular tests based on new nextgeneration sequencing have been discussed.

Folding and Binding Mechanisms of the SH2 Domain from Crkl.

SH2 domains are structural modules specialized in the recognition and binding of target sequences containing a phosphorylated tyrosine residue. They are mostly incorporated in the 3D structure of scaffolding proteins that represent fundamental regulators of several signaling pathways. Among those, Crkl plays key roles in cell physiology by mediating signals from a wide range of stimuli, and its overexpression is associated with several types of cancers. In myeloid cells expressing the oncogene BCR/ABL, one interactor of Crkl-SH2 is the focal adhesion protein Paxillin, and this interaction is crucial in leukemic transformation. In this work, we analyze both the folding pathway of Crkl-SH2 and its binding reaction with a peptide mimicking Paxillin, under different ionic strength and pH conditions, by using means of fluorescence spectroscopy. From a folding perspective, we demonstrate the presence of an intermediate along the reaction. Moreover, we underline the importance of the electrostatic interactions in the early event of recognition, occurring between the phosphorylated tyrosine of the Paxillin peptide and the charge residues of Crkl-SH2. Finally, we highlight a pivotal role of a highly conserved histidine residue in the stabilization of the binding complex. The experimental results are discussed in light of previous works on other SH2 domains.

Transcriptional factors control their diffusion on DNA by modulating their dynamics

Gene regulation requires transcription factors to efficiently recognize their target DNA site. Efficient recognition of target sequence is achieved by a facilitated diffusion process that combines 3D diffusion and 1D diffusive motions along the DNA, which are enabled by weak electrostatic (non-specific) interactions. The protein is presumed to scan the DNA while bound non-specifically, and lock into the specific binding site once it’s encountered. The mechanism by which a single domain protein alternates rapidly between these two binding modes is however unknown.

Determination of the Length of Target Recognition Sequence in sgRNA Required for CRISPR Interference

Determination of the Length of Target Recognition Sequence in sgRNA Required for CRISPR Interference

Sensitive electrochemical detection of oxytetracycline based on target triggered CHA and poly adenine assisted probe immobilization

Here, we developed a homogeneous electrochemical biosensor for the sensitive determination of antibiotic by the CHA reaction and the consecutive adenine mediated probe fixation. The binding of target to the target recognition sequences in the triple-helix DNA can release the trigger. It can initiate the catalytic hairpin assembly (CHA) to generate lots of mimic targets, which were labeled with electroactive substance ferrocene (Fc). Because the generated mimic target has consecutive sequence of adenines (PolyA), they can be self-assembled on the AuNPs modified electrode and finally realize electrochemical detection. Under optimal conditions, this developed biosensor achieved a satisfactory limit of detection of 0.089 nM (S/N = 3) and a linear range from 0.1 nM to 100 nM for sensitive detection of oxytetracycline with good specificity. The whole process is carried out in homogeneous solution, not only realizes signal amplification, but also avoids the complex modification process of electrode surface. Compared with some reported electrochemical sensors, the method is easier to operate and has good precision.

Genome Editing Revolution in Life Sciences

Programmable nucleases—ZFNs, TALENs and CRISPR-Cas9—have equipped scientists with an unprecedented ability to modify cells and organisms almost at will, with great implications across life sciences: biology, agriculture, ecology and medicine. Nucleases-based genome editing (aka gene editing) depends on cellular responses to a targeted double-strand break (DSB). The first truly targetable reagents were zinc finger nucleases (ZFNs) showing that arbitrary DNA sequences within a mammalian genome, could be addressed by protein engineering, ushering in the era of genome editing. ZFNs that are fusions of zinc finger proteins (ZFPs) and FokI cleavage domain, resulted from the basic research on Type IIs FokI restriction enzyme, which showed a bipartite structure with a separable DNA-binding domain and a non-specific cleavage domain. Studies on 3-finger ZFNs established that the preferred substrates were paired binding sites, which doubled the size of the target recognition sequence from 9 to 18 bp that is large enough to specify a unique genomic locus in plant and mammalian cells, including human cells. Subsequently, a ZFN-induced DSB was shown to stimulate homologous recombination in frog eggs. Transcription activator-like effector nucleases (TALENs) that are based on bacterial TALEs fused to FokI cleavage domain expanded the capability. ZFNs and TALENs have been successfully used to modify a multitude of recalcitrant organisms and cell types that were unapproachable previously attesting to the success of protein engineering, long before the arrival of CRISPR. The recent technique to deliver a targeted DSB to cellular genomes are RNA-guided nucleases as exemplified by the Type II prokaryotic CRISPR-Cas9 system. Unlike ZFNs and TALENs that use protein motifs for DNA sequence recognition, CRISPR-Cas9 depends on RNA-DNA recognition. The advantages of the CRISPR-Cas9 system, which include ease of RNA design for new targets and dependence on a single constant Cas9 protein, have led to its wide adoption by research labs around the world. The 2020 Nobel Prize for Chemistry was awarded to Jennifer Doudna and Emmanualle Charpentier for harnessing CRISPR-Cas9 system to provide a simplified technique for genome editing. The programmable nucleases have also been shown to cut at off-target sites with mutagenic consequences, which is a serious concern for human therapeutic applications. Therefore, applications of genome editing technologies to human therapeutics will ultimately depend on risk versus benefit analysis and informed consent