A Multivalent Structure-Specific RNA Binder with Extremely Stable Target Binding but Reduced Interaction with...

Area of Science:

• Biomolecular interactions
• Biosensing technologies
• Nucleic acid analysis

Background:

• Multivalent interactions enhance binding affinity for biosensing.
• A key challenge is increased nonspecific binding with multivalent probes.

Purpose of the Study:

• To engineer charge-modified PAZ probes with varying valencies (2-mer, 4-mer, 24-mer) using avidin proteins.
• To investigate the impact of charge engineering on specific and nonspecific RNA binding.
• To develop a highly sensitive and specific biosensor for microRNA detection.

Main Methods:

• Construction of multivalent PAZ probes using 2-, 4-, and 24-meric avidin proteins.
• Charge engineering of PAZ and avidin scaffolds.
• Evaluation of binding stability and specificity to structured target RNA and nonspecific RNA.
• Surface-enhanced Raman spectroscopy (SERS) for microRNA detection.

Main Results:

• Increased valency significantly enhanced PAZ probe binding stability to structured target RNA.
• Charge engineering on PAZ and avidin scaffolds reduced nonspecific RNA binding below monomer levels.
• The optimized 24-meric PAZ probe exhibited nearly irreversible binding to target RNA with negligible nonspecific binding.
• Attomolar detection of intact microRNAs was achieved using the ultra-specific 24-meric PAZ probe via SERS.

Conclusions:

• Multivalent interactions, when optimized with charge engineering, can overcome the challenge of nonspecific binding in biosensing.
• The developed ultra-specific 24-meric PAZ probe represents a significant advancement in sensitive and selective microRNA detection.
• This strategy holds promise for developing next-generation biosensors for various biomolecules.