Nucleic Acid Molecular Systems for In Vitro Detection of Biomolecules

Abstract

Molecular systems composed of information-rich nucleic acids have emerged as one of the most robust materials due to their programmability, editability, and designability. Among their various applications, the specific and sensitive in vitro detection of biomolecules for the purpose of disease diagnosis has attracted increasing attention from both fundamental and translational researchers. In this perspective, we introduce the basic design principles for nucleic acid molecular systems toward in vitro detection of biomolecules, accompanied by representative examples from reported works. The perspective concludes with perspectives and outlooks to tackle a variety of technical hurdles for the development and practical translation of nucleic acid molecular systems for biomolecule detection.

Introduction

Biomolecules including nucleic acids, proteins, and metabolic small molecules participate in basically all biochemical processes, which could serve as excellent indicators of the physiological and pathological states of the host body. Therefore, in vitro detection of biomolecules of interest has become an important aspect for accurate and comprehensive diagnosis of various diseases. There is an urgent need to develop sensitive, specific, portable, and cost-effective detection methods for biomolecules toward clinical translational applications.¹

Nucleic acids, the genetic blueprint for all known living organisms on Earth, exhibit a number of unique characteristics that make it nearly an ideal carrier for genetic information.² These characteristics include (1) sequence-specific base-pairing interactions (A pairs with T/U, C pairs with G); (2) well-defined and predictable nanoscale secondary structures (e.g., the right-handed B-form DNA duplex); and (3) template-dependent writability, readability, and editability by various nucleic acid enzymes. In addition to their role in genetics, it is not hard to realize that these properties would make nucleic acids among the best, if not the best, building blocks for the construction of designer artificial systems or synthetic materials with unprecedented programmable features. Polymerase chain reaction (PCR) represents one of the earliest and most elegant examples that design nucleic acid probes (i.e., primers) for the sequence-specific amplification/detection of targeting nucleic acids in an in vitro setting, which has since become the fundamental technique for molecular biology and for the biomedical field, in general.³ Fluorescence in situ hybridization (FISH) is yet another elegant example that utilizes fluorophore-labeled nucleic acid probes for sequence-specific detection and imaging of biological nucleic acids in cells or tissues.⁴ The field of DNA nanotechnology was founded by Nadrian Seeman in the early 1980s, where DNA (or RNA) molecules are de novo designed to self-assemble into designer nanoscale structures following prescribed sequence-specific interactions.⁵ Self-assembled nucleic acid structures exhibit unparallel properties such as precise geometry (i.e., size, shape) and addressability (placement of a certain molecule or material) that make them an excellent platform for constructing robust and versatile molecular detection systems.⁶

In the following section, nucleic acid-based molecular systems for the in vitro detection of biomolecules are summarized and discussed.

In Vitro Detection of Biomolecules

The workflow of using nucleic acid molecular systems for biomolecule detection is illustrated in Figure 1. Biomolecules from various biofluids (e.g., blood, urine, tears, saliva, nasal mucus, etc.) are extracted, amplified, and purified. For certain robust detection systems, this step may be skipped.⁷ Second, biomolecules are subject to incubation with nucleic acid systems for molecular recognition and detection. Signals transduced from the detection system may then be detected and recorded to evaluate the presence of specific biomolecules in a qualitative and/or quantitative manner. Depending on the nature of the output signal and on the specific requirements for analysis, a device may or may not be necessarily needed.

Figure 1.

Brief workflow using nucleic acid molecular systems for the in vitro detection of biomolecules from various biofluids.

Detection of Nucleic Acids

Sequence-specific base-pairing between nucleic acid detection probes and target nucleic acids is a universally used sensing mechanism due to its specificity, scalability, compatibility, and ease of design. For instance, PCR employs primers to specifically bind to targeting sequences of nucleic acids to achieve exponential amplification by using nucleic acid polymerases. In this section, we focused on the introduction of PCR-free nucleic acid detection systems. Toehold-mediated strand displacement (TMSD) is a commonly used strategy for detecting nucleic acids of specific sequences, where a single-stranded overhang serves as a toehold to initiate binding to target molecules which subsequently displaces the partially bound molecule (Figure 2a(i)).⁸ This displacement reaction leads to the release of a nucleic acid molecule that may serve as a transducer for generating output signals. Strand displacement may also integrate with biochemical machineries for nucleic acid detection. For example, a molecularly triggerable riboswitch is de novo designed to detect a target RNA molecule.⁹ The binding of target RNA onto the riboswitch recruits ribosome and initiates the synthesis of functional proteins that is compatible with fluorescent or colorimetric assays (Figure 2a(ii)). The CRISPR-Cas molecular system is one cutting-edge technique for nucleic acid detection.¹⁰ crRNA can specifically recognize and bind to target DNA/RNA, double-stranded or single-stranded, then activates the nuclease activity of Cas proteins. Cleavage of a nucleic acid reporter leads to the output of signals that may be detected by instruments or directly visualized by the naked eye (Figure 2a(iii)).

Figure 2.

Nucleic acid molecular system designs for the in vitro detection of biomolecules. (a) Detection of nucleic acids. (i) DNA probes for sequence-specific detection of nucleic acids based on toehold-mediated strand displacement reaction. (ii) Molecular triggerable riboswitch for nucleic acid detection. (iii) Sequence-specific nucleic acid recognition and detection platform with CRISPR-Cas systems. (b) Detection of proteins. (i) Nucleic acid aptamer-mediated detection of proteins. (ii) Nucleic acid–antibody chimera for protein detection via proximity extension assay. (iii) Protein detection by framework nucleic acid structures. (c) Detection of small molecules. (i) Nucleic acid aptamer-mediated detection of small molecules. (ii) Target small molecule serves as allosteric factor for turning on transcription. (iii) DNA origami templated metamolecule for small molecule detection by surface-enhanced Raman scattering.

Detection of Proteins

Nucleic acid aptamers are a group of single-stranded RNA or DNA molecules of known sequences that are able to specifically recognize and bind to target proteins (or small molecules), which are either naturally presented or in vitro selected.¹¹ To construct a nucleic acid molecular system for protein detection, the aptamer may first partially pair with another nucleic acid molecule (Figure 2b(i)). Binding to the target protein induces conformational change of the aptamer and subsequent displacement of the other nucleic acid molecule for signal output.¹² Nucleic acids may integrate with antibodies to build nucleic acid–antibody chimera detection systems (Figure 2b(ii)). For instance, single-stranded nucleic acids of barcode sequences can be conjugated onto antibodies. Recognition of an antigen by multiple antibodies leads to proximity extension of conjugated nucleic acids, which may then be sequenced to determine the presence and/or quantity of this specific target protein.¹³ We and others found that framework nucleic acid structures are able to interact and then capture serum proteins. Comprehensive investigation revealed that the profile of captured proteins was highly dependent on the physicochemical properties of nucleic acid structures and on the pathological states of serum donors.¹⁴ Based on this, specific diagnosis of patients with prostate cancer was achieved (Figure 2b(iii)).

Detection of Small Molecules

The same as with protein detection, the sensing module within the nucleic acid molecular system requires a molecular ligand that can interact with the targeting small molecules. As discussed above, aptamers are also capable of specifically binding to small molecules. Therefore, a straightforward system consisting of an aptamer and another nucleic acid molecule may be designed. Conformational change of aptamers upon small molecule binding has the other nucleic acid molecule being displaced and released to generate signals that can be detected (Figure 2c(i)). Allosteric transcription factors are a class of regulation proteins that are able to sense and cooperate with small molecules for mediating the transcription of genes. This molecular system may also work in in vitro setting to realize specific detection of targeting small molecules (Figure 2c(ii)).¹⁵ Artificial plasmonic metamolecules of strong surface-enhanced Raman scattering (SERS) properties may be readily assembled on DNA origami templates. The adsorption of targeting small molecules onto the plasmonic metamolecules induced significant changes in the SERS signal (Figure 2c(iii)).¹⁶

Perspectives and Outlooks

Remarkable progress has been made in nucleic acid molecular systems toward biomolecule detection applications in the past decade. Nevertheless, a variety of technical hurdles remain to be tackled in order to promote their translation to real clinical or field applications.

Sensitivity and Specificity

Sensitivity and specificity are among the most critical performance parameters of molecular detection systems. Amplification-based methods, such as PCR, or isothermal amplification (e.g., LAMP, RPA) are commonly used to reach high sensitivity. PCR, which uses polymerase to amplify the target nucleic acids, is the gold standard for clinical detection given its reliable and sensitive properties. Nevertheless, PCR requires trained personnel, expensive equipment, and a sterile environment, which impedes its widespread use. In addition, efficient amplification methods are vulnerable to contamination-induced (e.g., aerosol) false positive results or cause environmental contaminations due to amplicon leakage. A sealed, all-in-one reaction device (e.g., microfluidic chip) may alleviate contamination and leakage issues. Besides, amplification bias and difficulty in amplifying GC-rich regions are other challenges along with PCR. On the other hand, amplification-free methods may result in an irreproducible result due to relatively low sensitivity and complicated setups. Therefore, there is a balance to find within the detection system to achieve high sensitivity, specificity, and reliability. Although the sensitivity and specificity of a detection system are largely determined by the detection ligand (e.g., aptamer, antibody), spatial distribution of ligands within assembled molecular systems may also significantly affect the interactions between ligands and targeting molecules.¹⁷ Self-assembled nucleic acid platforms offer unprecedented capability on placing guest molecules of defined numbers and spatial locations. Several reports have demonstrated that a multivalent aptamer system may be used to capture circulating tumor cells or extracellular vesicles or to block viral particles, which are important for disease diagnosis and therapy. Therefore, we propose that one may rationally design and modulate parameters like the ligand number, composition, and spatial pattern to build molecular detection systems of tunable sensitivity and specificity against targeting biomolecules (Figure 3a).

Figure 3.

Perspectives and outlooks for the practical translation of nucleic acid-based biomolecule detection systems. (a) Enhance the sensitivity and specificity by harvesting the designability of nucleic acid molecular systems. (b) Enable multiplex detection capability on nucleic acid molecular system. (c) Integrate nucleic acid molecular system into an all-in-one test kit.

Multiplexing Capability

The capability of sensing and detecting various distinct biomolecules simultaneously will largely expand the application scenarios of molecular detection systems.¹⁸ To enable multiplexing capability, nucleic acid platforms may be readily used to integrate multiple detecting ligands into one sensing module (Figure 3b). After sensing the target biomolecules, the signals may be transduced to the classifying module for signal classification and reporting of detection results. The programmability of nucleic acid platforms enables orthogonal and synergistic operations of different detection pathways within the same molecular system that may be readily harvested for multiplexing detection. The major challenge for multiplexing detection is cross-talk between different targets and sensing modules, which would induce interference and difficulty for signal interpretation. To tackle this challenge, orthogonality and specificity of molecular detection systems are highly demanded. Nucleic acids are arguably the best molecules that can be designed to achieve excellent orthogonality and specificity, attributed to the high design capability of nucleic acid sequences and the associated sequence-specific molecular interactions. For instance, a pool of CRISPR-Cas molecular systems may be designed to detect four different nucleic acid targets at the same time by employing the orthogonality of crRNAs and the difference in collateral cleavage preference of CRISPR-Cas enzymes.¹⁹ In addition, orthogonality, specificity, and high throughput may be readily achieved by combining the detection systems with nucleic acid sequencing technologies.²⁰

Integratability

In many application scenarios of molecular detection systems such as for SARS-CoV-2 nucleic acid diagnosis,²¹ there is a practical and urgent need to reduce sample handling steps for the sake of avoiding samples being contaminated or the leakage of infectious materials into the environment or for shortening the test time. The nucleic acid platform exhibits robust versatility and compatibility to transform traditional serial sample handling procedures into a one-step test protocol by integrating everything all together including amplification, sensing, transducing, and signal reporting (Figure 3c). For example, nanostructure-based electrochemical miRNA quantification has been reported as a potential point-of-care platform.²² A wearable aptamer-based detection device has been reported for continuous monitoring of therapeutic drug concentrations in vivo.²³ An all-in-one test kit would have enormous practical application potentials in clinical settings or in fields demanding point-of-care testing.

In summary, molecular detection systems consisting of nucleic acids have demonstrated unparalleled capability and versatility for the in vitro detection of biomolecules in laboratories, which will soon be translated into bedside and field applications to largely contribute to and benefit human healthcare.

Author Contributions

CRediT: Donglei Yang conceptualization (supporting), writing-original draft (lead), writing-review & editing (lead); Lijiao Yang conceptualization (supporting), writing-original draft (supporting), writing-review & editing (supporting); Pengfei Wang conceptualization (lead), funding acquisition (lead), supervision (lead), writing-review & editing (lead).

The authors declare no competing financial interest.