RNA structure analysis often requires distinguishing single-stranded regions, double-stranded regions, stacked regions, and differences in structural accessibility. RNase T1, RNase A, RNase V1, and S1 nuclease can be used as structural probes. By selectively cleaving different conformational or base environments, they convert information on RNA secondary structure, local folding, protein-binding sites, and conformational changes into detectable fragment patterns.

Keywords: RNA structure analysis; nuclease probes; RNase T1; RNase A; RNase V1; S1 nuclease; RNA secondary structure; RNA footprinting; structural probes; RNA nuclease digestion

1 Core Logic of Nuclease Probe Technology

1.1 Structural accessibility and cleavage selectivity

Nuclease probe technology uses the selectivity of different nucleases toward RNA conformation, base type, or strand state to convert RNA structural information into differences in cleavage sites. Single-stranded regions are usually more readily cleaved by RNase T1, RNase A, or S1 nuclease, whereas double-stranded or stably stacked regions are more readily recognized by RNase V1. If a site is readily cleaved in free RNA but becomes resistant after protein binding, ligand binding, or changes in ionic conditions, it usually suggests protection, enhanced folding, or reduced accessibility in that region.

1.2 Applicable questions

(1) Secondary structure validation

Stem-loops, internal loops, bulges, and single-stranded linker regions predicted from sequence can be cross-validated using single-strand-specific and double-strand/stacking-specific nucleases. RNase T1, RNase A, and S1 are more suitable for locating open regions, whereas RNase V1 is more suitable for validating stem regions or local stacked structures.

(2) Conformational change analysis

RNA can undergo local structural rearrangement in response to Mg²⁺, ligands, temperature, pH, or protein binding. Changes in nuclease cleavage patterns can reflect regional protection, unfolding, stem formation, or dynamic exposure.

(3) RNA-protein interaction mapping

In RNA footprinting, protein-binding regions may show reduced nuclease cleavage, whereas neighboring structural rearrangement regions may show enhanced cleavage. Such results should be interpreted together with no-protein controls, protein gradients, mutant RNA, and independent binding assays.

Table 1 Recognition Features of Common RNA Structure Nuclease Probes

2 RNase T1: A Single-Stranded G-Site Structural Probe

2.1 Recognition features

RNase T1 primarily recognizes guanosine sites in single-stranded RNA and usually cleaves the phosphodiester bond after G. Its advantage is clear site selectivity, making it easier to map cleavage fragments to G positions in the RNA sequence. Its limitation is that it reports only G-related sites and does not cover A, C, or U regions.

2.2 Applicable scenarios

(1) Single-stranded G-site mapping

RNase T1 is suitable for validating the exposure status of G sites in hairpin loops, internal loops, bulges, and long single-stranded linker regions. If a G predicted to be in a single-stranded region shows strong cleavage, this generally supports accessibility at that site. If a G predicted to be in a double-stranded stem shows clear cleavage, local breathing, mismatch, dynamic unwinding, or instability of the predicted structure should be considered.

(2) Comparison of folding conditions

After the addition of Mg²⁺, salt, temperature changes, or ligands, changes in G-site cleavage intensity can reflect altered local accessibility. Reduced cleavage may indicate enhanced folding or binding protection, whereas increased cleavage may indicate structural opening or local instability.

(3) Sequencing ladders and structural map assistance

In RNA structure probing experiments, RNase T1 is often used to generate G-specific cleavage references to assist fragment positioning. For shorter RNAs, T1 patterns help rapidly establish correspondence between structural regions and sequence positions.

2.3 Interpretation points

RNase T1 signal intensity is affected by structural accessibility, G-site distribution, RNA concentration, and digestion time. High enzyme concentration or prolonged digestion may lead to overdigestion, making it difficult to distinguish primary cleavage events from secondary degradation. Structural analysis usually uses limited digestion conditions so that each RNA molecule undergoes only a small number of cleavage events on average.

3 RNase A: A Single-Stranded C/U-Site Probe

3.1 Recognition features

RNase A preferentially cleaves phosphodiester bonds after pyrimidine nucleotides in single-stranded RNA, mainly at C and U sites. It is highly active and stable and is suitable for identifying open single-stranded regions. However, because its cleavage sites are numerous, its pattern may be more complex than that of RNase T1.

3.2 Applicable scenarios

(1) C/U-rich loops and linker regions

When the single-stranded region of the target RNA contains many C or U residues, RNase A provides better coverage than RNase T1. If a loop, internal loop, or linker region shows continuous cleavage signals after RNase A treatment, this supports an open or dynamically exposed state.

(2) Comparison of single-stranded structural changes

After RNA binds protein, ligand, or metal ions, decreased cleavage at C/U sites may indicate local protection or structural stabilization. Increased cleavage may indicate conformational opening, increased strand exposure, or binding-induced local rearrangement.

(3) Complementary mapping with RNase T1

RNase T1 targets G, whereas RNase A targets C/U. Combining the two improves coverage of single-stranded regions. If a region is cleaved by both T1 and A, it more strongly supports an open single-stranded state. If only one enzyme produces a signal, base composition should be considered rather than directly interpreting it as a structural difference.

3.3 Interpretation points

RNase A is highly active, and overdigestion is the most common problem in experiments. Excess RNase A can rapidly degrade RNA, causing overly short fragments, loss of structural information, and increased background. When used as a structural probe, limited digestion conditions should be established through enzyme concentration and time gradients.

4 RNase V1: A Double-Stranded and Stacked-Structure Probe

4.1 Recognition features

RNase V1 preferentially cleaves double-stranded RNA, RNA-DNA hybrid duplexes, or regions of single-stranded RNA with stable stacked conformations. Unlike RNase T1 and RNase A, RNase V1 does not recognize specific bases but depends more on the spatial conformation and stacking state of the nucleic acid strand.

4.2 Applicable scenarios

(1) Stem validation

If a region predicted to be a double-stranded stem shows RNase V1 cleavage while being relatively resistant to RNase T1 or RNase A, this generally supports the formation of a stable double-stranded or stacked structure. If a predicted stem region lacks a V1 signal, a short stem length, high dynamics, local mismatch, or insufficient folding conditions should be considered.

(2) Identification of tightly folded regions

Some single-stranded RNAs do not form classical Watson-Crick duplexes but can still form V1-sensitive conformations through base stacking. In this case, a V1 signal should not be mechanically equated with a standard duplex; it should be interpreted as enhanced local stacking or compact conformation.

(3) Ligand- and protein-induced structural stabilization

Ligand binding, increased Mg²⁺, or protein binding may stabilize local RNA structures, appearing as enhanced RNase V1 cleavage. If single-strand nuclease signals decrease simultaneously, this supports a transition from an open state to a double-stranded/stacked state.

4.3 Interpretation points

RNase V1 results must be interpreted together with single-strand-specific nucleases. A V1 signal alone cannot fully prove duplex formation, because stably stacked single-stranded structures can also be cleaved. Conversely, lack of a V1 signal alone does not exclude duplex structure, because enzyme accessibility, secondary structure length, and reaction conditions can affect cleavage.

5 S1 Nuclease: A Probe for Single-Stranded Regions and Structural Gaps

5.1 Recognition features

S1 nuclease mainly cleaves single-stranded DNA and single-stranded RNA and is sensitive to single-stranded regions, gaps, loops, overhangs, and unpaired regions. It is not an RNA-specific nuclease, so in RNA structure studies it is mostly used for detecting exposed single-stranded regions or auxiliary validation of nucleic acid conformations.

5.2 Applicable scenarios

(1) Detection of exposed single-stranded regions

S1 nuclease is suitable for detecting obvious single-stranded regions, especially long single-stranded linkers, loops, or unstable local structures. Compared with RNase T1/RNase A, S1 does not depend on specific bases and can therefore provide structural information for regions lacking G or with low C/U content.

(2) Auxiliary analysis of RNA-DNA or DNA structures

In some nucleic acid structure studies, S1 can be used to detect exposed single-stranded regions or structural gaps in single-stranded DNA, RNA-DNA hybrid systems, or related structures. If both DNA and RNA are present in the system, the experimental target must be clearly defined; otherwise, the S1 signal may come from multiple single-stranded nucleic acid sources.

(3) Comparison of structural stability

Under different ionic, temperature, or ligand conditions, changes in S1 sensitivity can be used to evaluate the extent of single-strand exposure. A decrease in S1 signal usually suggests a more compact structure or fewer single-stranded regions, whereas an increase suggests local unfolding or increased single-strand exposure.

5.3 Interpretation points

S1 nuclease commonly works under mildly acidic conditions with Zn²⁺, and its reaction conditions may alter the folding state of some RNAs. Therefore, when S1 is used for RNA structure analysis, untreated controls, enzyme concentration gradients, and reaction condition controls are especially important to avoid mistaking condition-induced conformational changes for intrinsic structural features.

Table 2 Application Boundaries of Four Nuclease Probes

6 Experimental Design for Nuclease Probing

6.1 RNA folding conditions

RNA structure is highly sensitive to ions, temperature, RNA concentration, and refolding procedures. Before structural probing, RNA usually needs to undergo standardized heat denaturation, slow refolding, or equilibration under defined buffer conditions. If folding conditions differ between experimental groups, cleavage differences may originate from initial folding states rather than the treatment factor itself.

6.2 Principle of limited digestion

Nuclease probing emphasizes preservation of structural information, not complete digestion. Under limited digestion conditions, each RNA molecule undergoes only a small number of cleavage events on average, which better reflects original accessibility. Overdigestion produces secondary cleavage, overly short fragments, and increased background, making structural positioning difficult.

6.3 Time and enzyme gradients

Each nuclease has different activity, and different regions of the same RNA have different accessibility. Enzyme amount and time gradients should first be established to identify conditions that generate clear, reproducible cleavage without excessive degradation. RNase A requires especially strict optimization, whereas RNase V1 requires attention to Mg²⁺, buffer conditions, and RNA folding stability.

6.4 Fragment detection methods

Digestion products can be analyzed by denaturing PAGE, capillary electrophoresis, fluorescent primer extension, RT-stop detection, or sequencing. Short RNAs can be directly analyzed by denaturing PAGE. Complex RNAs or transcriptome-wide structural studies are more suitable for combination with high-throughput sequencing or structure-sequencing methods.

Table 3 Key Control Points in Nuclease Probe Experiments

7 Selection Strategies for Different Research Scenarios

7.1 Routine RNA secondary structure validation

Routine secondary structure validation can use a combination of RNase T1, RNase A, and RNase V1. T1 and A are used to locate open single-stranded regions, while V1 is used to confirm double-stranded or stacked regions. If the target RNA has few G sites, RNase A or S1 can be added. If dense C/U sites make the pattern too complex, RNase A concentration can be reduced and T1 used as the main single-strand probe.

7.2 RNA-protein interaction footprinting

RNA-protein interaction studies should compare the nuclease digestion patterns of free RNA and protein-bound RNA. Binding regions usually show nuclease protection, whereas structural rearrangement regions may show enhanced cleavage. To avoid broad false protection caused by protein obstruction of enzyme diffusion, protein concentration gradients, nonspecific protein controls, mutant RNA, and binding assays should be included.

7.3 Ligand-induced RNA conformational changes

In studies of riboswitches, aptamers, RNA drug targets, or small-molecule binding, nuclease probes can be used to observe ligand-induced structural stabilization. If RNase V1 signals increase after ligand addition while T1/A/S1 signals decrease, this usually suggests a more compact local structure. If single-strand signals increase, ligand-induced conformational release or local rearrangement may be indicated.

7.4 RNA stability and degradation sensitivity analysis

Different RNA folding states affect nuclease sensitivity. Stable stems, protein-bound regions, or areas near modification sites often show cleavage resistance, whereas open single-stranded, disordered, or structurally defective regions are more readily cleaved. This type of analysis should avoid directly equating nuclease sensitivity with intracellular degradation rate, because RNA-binding proteins, modifications, and subcellular localization also exist in cells.

Table 4 Recommended Nuclease Combinations for Different Experimental Purposes

8 Product Selection for RNA Structure Analysis and Nuclease Probe Experiments

Table 5 Product Selection for RNA Structure Analysis and Nuclease Probe Experiments

9 Result Interpretation and Common Pitfalls

9.1 A single nuclease cannot independently define structure

RNase T1 and RNase A signals indicate single-stranded accessibility but cannot exclude local dynamic breathing. RNase V1 signals indicate a double-stranded or stacked tendency but do not prove a standard Watson-Crick duplex. S1 signals indicate single-strand exposure but may be affected by reaction conditions. Reliable structural conclusions require multi-enzyme integration and consistency with structural models.

9.2 Enhanced cleavage does not necessarily mean complete structural opening

Enhanced cleavage may result from local unfolding, increased conformational fluctuation, protein-induced exposure of neighboring regions, or improved enzyme accessibility. Interpreting enhancement at a single site as opening of an entire region can lead to overinterpretation. Neighboring sites, opposite-type probes, and structural predictions should be considered together.

9.3 Cleavage resistance does not necessarily indicate direct protein binding

Protein-binding regions can show cleavage resistance, but enhanced RNA folding, steric shielding, changes in ionic conditions, or reduced enzyme activity can also cause resistance. In footprinting, protein gradients, mutants, competitive binding, and independent binding assays should be used to confirm direct binding sites.

9.4 Reaction buffers may alter RNA structure

Different nucleases have different optimal conditions, and Mg²⁺, Zn²⁺, pH, and salt concentration can all alter RNA folding. When comparing results from different nucleases, folding conditions should be controlled as consistently as possible, and interpretive boundaries caused by differences in reaction conditions should be recognized.

Table 6 Common Patterns and Interpretation of Nuclease Probe Results

10 Common Questions

10.1 RNase T1 and RNase A are both single-strand probes. How should they be selected?

RNase T1 mainly reports single-stranded G sites and provides clearer patterns, making it suitable for site positioning. RNase A mainly reports single-stranded C/U sites and provides broader coverage but is more prone to overdigestion. If the target RNA has few G sites, RNase A can be added. If dense C/U sites make the pattern complex, RNase A dosage can be reduced and T1 used for assisted positioning.

10.2 Does an RNase V1-cleaved region necessarily represent double-stranded RNA?

Not necessarily. RNase V1 prefers double-stranded or stably stacked structures, and some strongly stacked single-stranded regions may also be cleaved. Therefore, V1 signals should be interpreted together with T1, A, or S1 results and should not be used alone as evidence of a standard duplex.

10.3 Is S1 nuclease suitable as an RNA structure probe?

It can be used to detect single-stranded regions and structural gaps, but S1 is not RNA-specific, and its reaction conditions may affect RNA folding. If DNA or RNA-DNA hybrid structures are present in the system, the source of the S1 signal should be clarified and appropriate controls should be included.

10.4 Why do nuclease probe experiments require limited digestion?

Limited digestion preserves information on the original structural accessibility. Complete digestion causes multiple cleavage events in RNA molecules, resulting in overly short fragments and difficulty in identifying primary cleavage sites, which substantially reduces structural analysis value.

10.5 In RNA-protein footprinting, is a cleavage-resistant region necessarily a protein-binding site?

Not necessarily. A cleavage-resistant region may represent direct protein binding, but it may also result from protein-induced RNA folding enhancement or steric shielding. Protein concentration gradients, mutant RNA, EMSA, CLIP, or other binding assays should be used for confirmation.

10.6 What does increased RNase V1 signal after ligand addition indicate?

It usually suggests enhanced local double-stranded or stacked RNA structure, but it may also reflect ligand-induced changes in enzyme accessibility. If single-strand nuclease signals decrease simultaneously and the change increases with ligand concentration, ligand-induced structural stabilization is more strongly supported.

10.7 Can nuclease probing replace SHAPE or DMS?

Not completely. Nuclease probing is suitable for rapidly distinguishing single-stranded, double-stranded, and accessibility changes. SHAPE and DMS are more suitable for site-level chemical modification and structural modeling. In complex RNA structure studies, the two approaches can be complementary.

The value of nuclease probe technology lies in using the complementary selectivity of multiple enzymes to analyze RNA structural states. RNase T1 and RNase A emphasize single-stranded sites, RNase V1 emphasizes double-stranded or stacked regions, and S1 nuclease supplements sequence-independent single-strand detection. Only by integrating multi-enzyme patterns, folding conditions, functional binding assays, and structural predictions can local RNA structures and their regulatory changes be interpreted more accurately.

For more related articles, please see below:

[1] RNA footprinting and modification interference analysis experiments