Ionic liquid-caged nucleic acids enable active folding-based molecular recognition with hydrolysis resistance.

Beyond storage and transmission of genetic information in cellular life, nucleic acids can perform diverse interesting functions, including specific target recognition and biochemical reaction acceleration; the versatile biopolymers, however, are acutely vulnerable to hydrolysis-driven degradation. Here, we demonstrate that the cage effect of choline dihydrogen phosphate permits active folding of nucleic acids like water, but prevents their phosphodiester hydrolysis unlike water. The choline-based ionic liquid not only serves as a universal inhibitor of nucleases, exceptionally extending half-lives of nucleic acids up to 6 500 000 times, but highly useful tasks of nucleic acids (e.g. mRNA detection of molecular beacons, ligand recognition of aptamers, and transesterification reaction of ribozymes) can be also conducted with well-conserved affinities and specificities. As liberated from the function loss and degradation risk, the presence of undesired and unknown nucleases does not undermine desired molecular functions of nucleic acids without hydrolysis artifacts even in nuclease cocktails and human saliva.

A unified computational view of DNA duplex, triplex, quadruplex and their donor-acceptor interactions.

DNA can assume various structures as a result of interactions at atomic and molecular levels (e.g., hydrogen bonds, π-π stacking interactions, and electrostatic potentials), so understanding of the consequences of these interactions could guide development of ways to produce elaborate programmable DNA for applications in bio- and nanotechnology. We conducted advanced ab initio calculations to investigate nucleobase model structures by componentizing their donor-acceptor interactions. By unifying computational conditions, we compared the independent interactions of DNA duplexes, triplexes, and quadruplexes, which led us to evaluate a stability trend among Watson-Crick and Hoogsteen base pairing, stacking, and even ion binding. For a realistic solution-like environment, the influence of water molecules was carefully considered, and the potassium-ion preference of G-quadruplex was first analyzed at an ab initio level by considering both base-base and ion-water interactions. We devised new structure factors including hydrogen bond length, glycosidic vector angle, and twist angle, which were highly effective for comparison between computationally-predicted and experimentally-determined structures; we clarified the function of phosphate backbone during nucleobase ordering. The simulated tendency of net interaction energies agreed well with that of real world, and this agreement validates the potential of ab initio study to guide programming of complicated DNA constructs.

In vitro selection of ribozyme ligases that use prebiotically plausible 2-aminoimidazole-activated substrates.

The hypothesized central role of RNA in the origin of life suggests that RNA propagation predated the advent of complex protein enzymes. A critical step of RNA replication is the template-directed synthesis of a complementary strand. Two experimental approaches have been extensively explored in the pursuit of demonstrating protein-free RNA synthesis: template-directed nonenzymatic RNA polymerization using intrinsically reactive monomers and ribozyme-catalyzed polymerization using more stable substrates such as biological 5'-triphosphates. Despite significant progress in both approaches in recent years, the assembly and copying of functional RNA sequences under prebiotic conditions remains a challenge. Here, we explore an alternative approach to RNA-templated RNA copying that combines ribozyme catalysis with RNA substrates activated with a prebiotically plausible leaving group, 2-aminoimidazole (2AI). We applied in vitro selection to identify ligase ribozymes that catalyze phosphodiester bond formation between a template-bound primer and a phosphor-imidazolide-activated oligomer. Sequencing revealed the progressive enrichment of 10 abundant sequences from a random sequence pool. Ligase activity was detected in all 10 RNA sequences; all required activation of the ligator with 2AI and generated a 3'-5' phosphodiester bond. We propose that ribozyme catalysis of phosphodiester bond formation using intrinsically reactive RNA substrates, such as imidazolides, could have been an evolutionary step connecting purely nonenzymatic to ribozyme-catalyzed RNA template copying during the origin of life.

Live Streaming of a Single Cell's Life over a Local pH-Monitoring Nanowire Waveguide.

Spatiotemporal pH monitoring of single living cells across rigid cell and organelle membranes has been challenging, despite its significance in understanding cellular heterogeneity. Here, we developed a mechanically robust yet tolerably thin nanowire waveguide that enables in situ monitoring of pH dynamics at desired cellular compartments via direct optical communication. By chemically labeling fluorescein at one end of a poly(vinylbenzyl azide) nanowire, we continuously monitored pH variations of different compartments inside a living cell, successfully observing organelle-exclusive pH homeostasis and stimuli-selective pH regulations. Importantly, it was demonstrated for the first time that, during the mammalian cell cycle, the nucleus displays pH homeostasis in interphase but a tidal pH curve in the mitotic phase, implying the existence of independent pH-regulating activities by the nuclear envelope. The rapid and accurate local pH-reporting capability of our nanowire waveguide would be highly valuable for investigating cellular behaviors under diverse biological situations in living cells.

Systematic Combination of Oligonucleotides and Synthetic Polymers for Advanced Therapeutic Applications.

The potential of oligonucleotides is exceptional in therapeutics because of their high safety, potency, and specificity compared to conventional therapeutic agents. However, many obstacles, such as low in vivo stability and poor cellular uptake, have hampered their clinical success. Use of polymeric carriers can be an effective approach for overcoming the biological barriers and thereby maximizing the therapeutic efficacy of the oligonucleotides due to the availability of highly tunable synthesis and functional modification of various polymers. As loaded in the polymeric carriers, the therapeutic oligonucleotides, such as antisense oligonucleotides, small interfering RNAs, microRNAs, and even messenger RNAs, become nuclease-resistant by bypassing renal filtration and can be efficiently internalized into disease cells. In this review, we introduced a variety of systematic combinations between the therapeutic oligonucleotides and the synthetic polymers, including the uses of highly functionalized polymers responding to a wide range of endogenous and exogenous stimuli for spatiotemporal control of oligonucleotide release. We also presented intriguing characteristics of oligonucleotides suitable for targeted therapy and immunotherapy, which can be fully supported by versatile polymeric carriers.

Ultra-Stable Freestanding Lipid Membrane Array: Direct Visualization of Dynamic Membrane Remodeling with Cholesterol Transport and Enzymatic Reactions.

Cell membranes actively change their local compositions, serving essential biological processes such as cellular signaling and endocytosis. Although membrane dynamics is vital in the cellular functions, the complexity of natural membranes has made its fundamental understanding and systematic assessment difficult. Here, a powerful artificial membrane system is developed for real-time visualization of the spatiotemporal dynamics of membrane remodeling. Through well-defined air/oil/water interfaces on grid holes, tens of planar lipid bilayer membranes are easily created, and their reproducibility, controllability, and generality are highlighted. The freestanding membranes are large but also highly stable, facilitating direct long-term monitoring of dynamic membrane reconstitution caused by external stimuli. As an example to demonstrate the superiority of this membrane system, the effect of cholesterol trafficking, which significantly affects biophysical properties of cell membranes, is investigated at different membrane compositions. Cholesterol transport into and out of the membranes at different rates causes anomalous lipid arrangements through cholesterol-mediated phase transitions and decomposition, which have never been witnessed before. Furthermore, enzyme-induced membrane dynamics is successfully shown in this platform; sphingomyelinases locally generate asymmetry between two membrane leaflets. This technique is broadly applicable for exploring the membrane heterogeneity under various membrane-based reactions, providing valuable insight into the membrane dynamics.

Synthesis of DNA-templated copper nanoparticles with enhanced fluorescence stability for cellular imaging.

Fluorescence of DNA-templated copper nanoparticles (DNA-CuNPs) is not stable over time which limits applications in cellular imaging. This is due to the presence of oxygen during synthesis which oxidizes Cu(0) to Cu(II) and also produces the free hydroxyl radical. The authors have prepared DNA-CuNPs with enhanced temporal stability of fluorescence by optimizing the reaction conditions so as to minimize the deleterious effects of oxygen. The operational lifetime of DNA-CuNPs was increased from 25 min to 200 min. Fluorescence spectra of DNA-CuNPs in optimized condition show an emission peak at 650 nm when excited at 340 nm. DNA-CuNPs synthesized in this manner were used for cell imaging. As a proof of concept, the nucleus of a human colon cell line (HCT116) was stained. The method does not involve any chemicals other that copper sulfate and ascorbate. This new approach for generating DNA-CuNPs improves imaging of biological processes and provides a basis for developing other types of DNA-templated nanomaterials. Graphical abstract Schematic presentation of the formation of fluorescent DNA-templated copper nanoparticles (DNA-CuNPs). A large amount of ascorbate provides long operational lifetime for cellular imaging under the condition exposed to oxygen. *Asc- and **DHA stand for ascorbate and dehydroascorbic acid.

Structural Rationale for the Enhanced Catalysis of Nonenzymatic RNA Primer Extension by a Downstream Oligonucleotide.

Nonenzymatic RNA primer extension by activated mononucleotides has long served as a model for the study of prebiotic RNA copying. We have recently shown that the rate of primer extension is greatly enhanced by the formation of an imidazolium-bridged dinucleotide between the incoming monomer and a second, downstream activated monomer. However, the rate of primer extension is further enhanced if the downstream monomer is replaced by an activated oligonucleotide. Even an unactivated downstream oligonucleotide provides a modest enhancement in the rate of reaction of a primer with a single activated monomer. Here we study the mechanism of these effects through crystallographic studies of RNA complexes with the recently synthesized nonhydrolyzable substrate analog, guanosine 5'-(4-methylimidazolyl)-phosphonate (ICG). ICG mimics 2-methylimidazole activated guanosine-5'-phosphate (2-MeImpG), a commonly used substrate in nonenzymatic primer extension experiments. We present crystal structures of primer-template complexes with either one or two ICG residues bound downstream of a primer. In both cases, the aryl-phosphonate moiety of the ICG adjacent to the primer is disordered. To investigate the effect of a downstream oligonucleotide, we transcribed a short RNA oligonucleotide with either a 5'-ICG residue, a 5'-phosphate or a 5'-hydroxyl. We then determined crystal structures of primer-template complexes with a bound ICG monomer sandwiched between the primer and each of the three downstream oligonucleotides. Surprisingly, all three oligonucleotides rigidify the ICG monomer conformation and position it for attack by the primer 3'-hydroxyl. Furthermore, when GpppG, an analog of the imidazolium-bridged intermediate, is sandwiched between an upstream primer and a downstream helper oligonucleotide, or covalently linked to the 5'-end of the downstream oligonucleotide, the complex is better preorganized for primer extension than in the absence of a downstream oligonucleotide. Our results suggest that a downstream helper oligonucleotide contributes to the catalysis of primer extension by favoring a reactive conformation of the primer-template-intermediate complex.

In Vitro Selection of pH-Activated DNA Nanostructures.

We report the first in vitro selection of DNA nanostructures that switch their conformation when triggered by change in pH. Previously, most pH-active nanostructures were designed using known pH-active motifs, such as the i-motif or the triplex structure. In contrast, we performed de novo selections starting from a random library and generated nanostructures that can sequester and release Mipomersen, a clinically approved antisense DNA drug, in response to pH change. We demonstrate extraordinary pH-selectivity, releasing up to 714-fold more Mipomersen at pH 5.2 compared to pH 7.5. Interestingly, none of our nanostructures showed significant sequence similarity to known pH-sensitive motifs, suggesting that they may operate via novel structure-switching mechanisms. We believe our selection scheme is general and could be adopted for generating DNA nanostructures for many applications including drug delivery, sensors and pH-active surfaces.

Thousand-fold volumetric concentration of live cells with a recirculating acoustofluidic device.

The ability to concentrate cells from dilute samples into smaller volumes is an essential process step for most biological assays. Volumetric concentration is typically achieved via centrifugation, but this technique is not well suited for handling small number of cells, especially outside of the laboratory setting. In this work, we describe a novel device that combines acoustofluidics with a recirculating architecture to achieve >1000-fold enrichment of cells in a label-free manner, at high volumetric throughput (>500 µL min(-1)) and with high recovery (>98.7%). We demonstrate that our device can be used with a wide variety of different cell types and show that this concentration strategy does not affect cell viability. Importantly, our device could be readily adopted to serve as a "sample preparation" module that can be integrated with other microfluidic devices to allow analysis of dilute cellular samples in large volumes.

Array-based discovery of aptamer pairs.

Affinity reagent pairs that recognize distinct epitopes on a target protein can greatly improve the sensitivity and specificity of molecular detection. Importantly, such pairs can be conjugated to generate reagents that achieve two-site "bidentate" target recognition, with affinities greatly exceeding either monovalent component. DNA aptamers are especially well-suited for such constructs, because they can be linked via standard synthesis techniques without requiring chemical conjugation. Unfortunately, aptamer pairs are difficult to generate, primarily because conventional selection methods preferentially yield aptamers that recognize a dominant "hot spot" epitope. Our array-based discovery platform for multivalent aptamers (AD-MAP) overcomes this problem to achieve efficient discovery of aptamer pairs. We use microfluidic selection and high-throughput sequencing to obtain an enriched pool of aptamer sequences. Next, we synthesize a custom array based on these sequences, and perform parallel affinity measurements to identify the highest-affinity aptamer for the target protein. We use this aptamer to form complexes that block the primary binding site on the target, and then screen the same array with these complexes to identify aptamers that bind secondary epitopes. We used AD-MAP to discover DNA aptamer pairs that bind distinct sites on human angiopoietin-2 with high affinities, even in undiluted serum. To the best of our knowledge, this is the first work to discover new aptamer pairs using arrays. We subsequently conjugated these aptamers with a flexible linker to construct ultra-high-affinity bidentate reagents, with equilibrium dissociation constants as low as 97 pM: >200-fold better than either component aptamer. Functional studies confirm that both aptamers critically contribute to this ultrahigh affinity, highlighting the promise of such reagents for research and clinical use.

Synthetic aptamer-polymer hybrid constructs for programmed drug delivery into specific target cells.

Viruses have evolved specialized mechanisms to efficiently transport nucleic acids and other biomolecules into specific host cells. They achieve this by performing a coordinated series of complex functions, resulting in delivery that is far more efficient than existing synthetic delivery mechanisms. Inspired by these natural systems, we describe a process for synthesizing chemically defined molecular constructs that likewise achieve targeted delivery through a series of coordinated functions. We employ an efficient "click chemistry" technique to synthesize aptamer-polymer hybrids (APHs), coupling cell-targeting aptamers to block copolymers that secure a therapeutic payload in an inactive state. Upon recognizing the targeted cell-surface marker, the APH enters the host cell via endocytosis, at which point the payload is triggered to be released into the cytoplasm. After visualizing this process with coumarin dye, we demonstrate targeted killing of tumor cells with doxorubicin. Importantly, this process can be generalized to yield APHs that specifically target different surface markers.

Selection of phage-displayed peptides on live adherent cells in microfluidic channels.

Affinity reagents that bind to specific molecular targets are an essential tool for both diagnostics and targeted therapeutics. There is a particular need for advanced technologies for the generation of reagents that specifically target cell-surface markers, because transmembrane proteins are notoriously difficult to express in recombinant form. We have previously shown that microfluidics offers many advantages for generating affinity reagents against purified protein targets, and we have now significantly extended this approach to achieve successful in vitro selection of T7 phage-displayed peptides that recognize markers expressed on live, adherent cells within a microfluidic channel. As a model, we have targeted neuropilin-1 (NRP-1), a membrane-bound receptor expressed at the surface of human prostate carcinoma cells that plays central roles in angiogenesis, cell migration, and invasion. We show that, compared to conventional biopanning methods, microfluidic selection enables more efficient discovery of peptides with higher affinity and specificity by providing controllable and reproducible means for applying stringent selection conditions against minimal amounts of target cells without loss. Using our microfluidic system, we isolate peptide sequences with superior binding affinity and specificity relative to the well known NRP-1-binding RPARPAR peptide. As such microfluidic systems can be used with a wide range of biocombinatorial libraries and tissue types, we believe that our method represents an effective approach toward efficient biomarker discovery from patient samples.

Aptasensor-encapsulating semi-permeable proteinosomes for direct target detection in non-treated biofluids.

Detecting biomarkers in biofluids directly without sample treatments makes molecular diagnostics faster and more efficient. Aptasensors, the nucleic acid-based molecular biosensors, can detect a wide range of target molecules, but their susceptibility to degradation and aggregation by nucleases and charged proteins, respectively, limits their direct use in clinical samples. In this work, we demonstrate that when aptasensors are encapsulated in proteinosomes, the protein-based liposome mimics, clinically important small molecules can be sensitively and selectively detected in non-treated specimens, such as 100 % unpurified serum. As serum albumin is used to form the membrane, the nanomeshed proteinosomes become semi-permeable and antifouling, which enables exclusive admission of small molecules while blocking unwanted large proteins. Consequently, the enclosed aptasensors can maintain close-to-optimal performance for target binding, and nucleolytic degradation and electrostatic aggregation are effectively suppressed. Three different structure-switching aptamers specific for estradiol, dopamine, and cocaine, respectively, are demonstrated to fully conserve their high affinities and specificities inside the microcapsules. The shielding effect of proteinosomes is indeed exceptional; the enclosed DNA aptasensors remain completely intact over 18 h in serum and even in an extremely concentrated DNase solution (1 mg/ml, ∼300,000× the serum level). Moreover, the proteinosome-mediated compartmentalization enables independent operation of multiple aptasensors in the same mixture. Hence, simultaneous real-time sensing of two different targets is demonstrated with different operation modes, 'recording' target appearance and 'reporting' target concentration changes. This work is the first demonstration of small-molecule-specific aptasensors operating with optimal performance in serum environments and will find promising applications in molecular diagnostics.

Arginine-Rich Cell-Penetrating Peptides Induce Lipid Rearrangements for Their Active Translocation across Laterally Heterogeneous Membranes.

Arginine-rich cell-penetrating peptides (CPPs) have emerged as valuable tools for the intracellular delivery of bioactive molecules, but their membrane perturbation during cell penetration is not fully understood. Here, nona-arginine (R9)-mediated membrane reorganization that facilitates the translocation of peptides across laterally heterogeneous membranes is directly visualized. The electrostatic binding of cationic R9 to anionic phosphatidylserine (PS)-enriched domains on a freestanding lipid bilayer induces lateral lipid rearrangements; in particular, in real-time it is observed that R9 fluidizes PS-rich liquid-ordered (Lo) domains into liquid-disordered (Ld) domains, resulting in the membrane permeabilization. The experiments with giant unilamellar vesicles (GUVs) confirm the preferential translocation of R9 through Ld domains without pore formation, even when Lo domains are more negatively charged. Indeed, whenever R9 comes into contact with negatively charged Lo domains, it dissolves the Lo domains first, promoting translocation across phase-separated membranes. Collectively, the findings imply that arginine-rich CPPs modulate lateral membrane heterogeneity, including membrane fluidization, as one of the fundamental processes for their effective cell penetration across densely packed lipid bilayers.

In vitro selection of a trans aptamer complex for target-responsive fluorescence activation.

BACKGROUND: Most biological molecular complexes consist of multiple functional domains, yet rationally constructing such multifunctional complexes is challenging. Aptamers, the nucleic acid-based functional molecules, can perform multiple tasks including target recognition, conformational changes, and enzymatic activities, while being chemically synthesizable and tunable, and thus provide a basis for engineering enhanced functionalities through combination of multiple units. However, the conventional approach of simply combining aptamer units in a serial manner is susceptible to undesired crosstalk or interference between the aptamer units and to false interactions with non-target molecules; besides, the approach would require additional mechanisms to separate the units if they are desired to function independently. It is clearly a challenge to develop multi-aptamer complexes that preserve independent functions of each unit while avoiding undesired interference and non-specific interactions. RESULTS: By directly in vitro selecting a 'trans' aptamer complex, we demonstrate that one aptamer unit ('utility module') can remain hidden or 'inactive' until a target analyte triggers the other unit ('sensing module') and separates the two aptamers. Since the operation of the utility module occurs free from the sensing module, unnecessary crosstalk between the two units can be avoided. Because the utility module is kept inactive until separated from the complex, non-specific interactions of the hidden module with noncognate targets can be naturally prevented. In our demonstration, the sensing module was selected to detect serotonin, a clinically important neurotransmitter, and the target-binding-induced structure-switching of the sensing module reveals and activates the utility module that turns on a fluorescence signal. The aptamer complex exhibited a moderately high affinity and an excellent specificity for serotonin with ∼16-fold discrimination against common neurotransmitter molecules, and displayed strong robustness to perturbations in the design, disallowing nonspecific reactions against various challenges. SIGNIFICANCE: This work represents the first example of a trans aptamer complex that was in vitro selected de novo. The trans aptamer complex selected by our strategy does not require chemical modifications or immediate optimization processes to function, because the complex is directly selected to perform desired functions. This strategy should be applicable to a wide range of functional nucleic acid moieties, which will open up diverse applications in biosensing and molecular therapeutics.

Senescent Astrocytes Derived from Human Pluripotent Stem Cells Reveal Age-Related Changes and Implications for Neurodegeneration.

Astrocytes play a crucial role in maintaining brain homeostasis by regulating synaptic activity, providing metabolic support to neurons, and modulating immune responses in the central nervous system (CNS). During aging, astrocytes undergo senescence with various changes that affect their function and frequently lead to neurodegeneration. This study presents the first evidence of senescent astrocytes derived from human pluripotent stem cells (hPSCs). These senescent hPSC-derived astrocytes exhibited altered cellular and nuclear morphologies, along with increased expression of senescence-associated markers. Additionally, nuclear localization of NFκB, telomere shortening, and frequent signs of DNA damage were observed in these cells. Furthermore, senescent astrocytes showed defects in various critical functions necessary for maintaining a healthy CNS environment, including a reduced ability to support neuronal survival and clear neurotransmitters, synaptic debris, and toxic protein aggregates. Altered structural dynamics and reduced mitochondrial function were also observed in senescent astrocytes. Notably, treating hPSC-derived senescent astrocytes with chemicals targeting reactive oxygen species or an enzyme that regulates mitochondrial function can reverse senescence phenotypes. Thus, this study offers a valuable cellular model that can be utilized to investigate the mechanisms of brain aging and may present new avenues for discovering innovative therapeutic approaches for neurodegenerative diseases.

Alveolar Microdynamics during Tidal Ventilation in Live Animals Imaged by SPring-8 Synchrotron.

It is self-evident that our chests expand and contract during breathing but, surprisingly, exactly how individual alveoli change shape over the respiratory cycle is still a matter of debate. Some argue that all the alveoli expand and contract rhythmically. Others claim that the lung volume change is due to groups of alveoli collapsing and reopening during ventilation. Although this question might seem to be an insignificant detail for healthy individuals, it might be a matter of life and death for patients with compromised lungs. Past analyses were based on static post-mortem preparations primarily due to technological limitations, and therefore, by definition, incapable of providing dynamic information. In contrast, this study provides the first comprehensive dynamic data on how the shape of the alveoli changes, and, further, provides valuable insights into the optimal lung volume for efficient gas exchange. It is concluded that alveolar micro-dynamics is nonlinear; and at medium lung volume, alveoli expand more than the ducts.

In vitro selection of structure-switching, self-reporting aptamers.

We describe an innovative selection approach to generate self-reporting aptamers (SRAs) capable of converting target-binding events into fluorescence readout without requiring additional modification, optimization, or the use of DNA helper strands. These aptamers contain a DNAzyme moiety that is initially maintained in an inactive conformation. Upon binding to their target, the aptamers undergo a structural switch that activates the DNAzyme, such that the binding event can be reported through significantly enhanced fluorescence produced by a specific stacking interaction between the active-conformation DNAzyme and a small molecule dye, N-methylmesoporphyrin IX. We demonstrate a purely in vitro selection-based approach for obtaining SRAs that function in both buffer and complex mixtures such as blood serum; after 15 rounds of selection with a structured DNA library, we were able to isolate SRAs that possess low nanomolar affinity and strong specificity for thrombin. Given ongoing progress in the engineering and characterization of functional DNA/RNA molecules, strategies such as ours have the potential to enable rapid, efficient, and economical isolation of nucleic acid molecules with diverse functionalities.

Quantitative selection of DNA aptamers through microfluidic selection and high-throughput sequencing.

We describe the integration of microfluidic selection with high-throughput DNA sequencing technology for rapid and efficient discovery of nucleic acid aptamers. The Quantitative Selection of Aptamers through Sequencing method tracks the copy number and enrichment-fold of more than 10 million individual sequences through multiple selection rounds, enabling the identification of high-affinity aptamers without the need for the pool to fully converge to a small number of sequences. Importantly, this method allows the discrimination of sequences that arise from experimental biases rather than true high-affinity target binding. As a demonstration, we have identified aptamers that specifically bind to PDGF-BB protein with K(d) < 3 nM within 3 rounds. Furthermore, we show that the aptamers identified by Quantitative Selection of Aptamers through Sequencing have approximately 3-8-fold higher affinity and approximately 2-4-fold higher specificity relative to those discovered through conventional cloning methods. Given that many biocombinatorial libraries are encoded with nucleic acids, we extrapolate that our method may be extended to other types of libraries for a range of molecular functions.