Modified i-Switch Nucleic Acid Complexes for Broad pH Reporting
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Solution Overview
Problem
Current pH-responsive i-switches have limited pH reporting capacity, primarily effective between pH 5.5 and 7.0, which does not cover the entire physiological range of intracellular organelles, and increasing cooperativity narrows the pH-sensitive regime, making it challenging to alter the pH midpoint without increasing cooperativity.
Innovation Solution
Development of nucleic acid complexes with modified cytosine residues, such as 5'-Bromocytosine and 5'-Methylcytosine, integrated into specific positions within the i-switch sequence to alter the pH midpoint of structural transitions while maintaining cooperativity, enabling pH reporting across a broader physiological range from pH 4.5 to 8.0.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If the number of cytosines in a stretch is increased to increase cooperativity, then the pH resolution is improved, but the pH sensitive regime is substantially narrowed
Solution Approach 1:
The patent applies parameter changes by substituting modified cytosine residues (5-methylcytosine, 5-bromocytosine, 5-propynylcytosine) for natural cytosine residues within the i-motif forming sequence. These chemical modifications alter the pKa values of the cytosine bases, thereby shifting the pH midpoint (pH1/2) of the conformational transition without increasing the number of cytosines. This resolves the contradiction by achieving pH resolution improvement through chemical parameter modification rather than increasing sequence length, thus maintaining a broad pH sensitive regime.
2Measurement precision
If the pH midpoint of the structural transition is altered by increasing cooperativity, then the pH reporting capacity is improved, but the overall pH sensitive regime is narrowed
Solution Approach 1:
The patent modifies the chemical parameters of the cytosine residues through substitution with modified bases (5-methylcytosine, 5-bromocytosine, 5-propynylcytosine). These modifications change the electronic properties and pKa values of the cytosine rings, enabling precise control over the pH midpoint of the i-motif transition. This approach achieves improved pH reporting capacity at desired midpoints without requiring increased cooperativity, thus preserving a broad pH sensitive regime.
Solution Approach 2:
The modified cytosine residues act as intermediaries that mediate the pH response of the i-motif structure. By introducing these modified bases at specific positions within the sequence, the patent fine-tunes the pH sensitivity and midpoint of the conformational transition. The modified cytosines serve as chemical mediators that translate pH changes into structural transitions at predetermined midpoints, enabling customized pH reporting ranges while maintaining broad adaptability.
3Adaptability or versatility
If modified cytosine residues are integrated into the i-switch sequence, then the pH midpoint is altered, but maintaining cooperativity and stability becomes challenging
Solution Approach 1:
The patent applies local quality by strategically positioning modified cytosine residues at specific locations within the i-motif sequence rather than uniformly distributing them. By placing modified bases at key positions (such as at the interfaces between stacked duplexes or at specific helical positions), the patent locally modulates the pH sensitivity and midpoint while preserving the overall cooperative folding behavior and structural stability of the i-motif. This localized modification approach allows precise pH midpoint control without compromising global stability.
Solution Approach 2:
The patent creates composite nucleic acid structures by combining modified cytosine residues with natural nucleotide bases within the same i-motif sequence. This composite approach allows the modified cytosines to provide targeted pH sensitivity adjustments while the natural bases maintain the fundamental structural framework and cooperative folding properties. The composite sequence design enables tuning of pH midpoint while preserving the reliability and stability characteristic of natural i-motifs.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
The modified i-switches demonstrate enhanced sensitivity and pH reporting capacity across the entire physiological range, maintaining cooperativity and stability, allowing for precise pH monitoring in various intracellular environments.
Implementation Method 1
a first label is capable of producing a signal, wherein the intensity of the signal varies as a function of the conformation of the nucleic acid complex
Implementation Method 2
Cytosine hemiprotonation drives i-motif formation. The pKa of Cytosine N3 is ̃4.5 and thus DNA4 i-motifs are maximally stabilized at approximately pH 5.0
Implementation Method 3
the pH responsive regime of i-motif based conformational switches may be tuned by changing the number of cytosines in a stretch, since cooperativity correlates directly with i-motif stability
Data Source
AI summary
Described herein are nucleic acid molecules and complexes useful as i-switch pH reporters that have increased sensitivities as a pH reporter and have alternate pH reporting capacity ranges. Aspects of the disclosure relate to a method for determining pH comprising providing a nucleic acid complex comprising: a first single-stranded nucleic acid molecule comprising the sequence CnXCnYCnZCn (SEQ ID NO. 6) wherein C is cytosine; X, Y and Z are each one or more of adenine, thymine, guanine, or combinations thereof; and n is greater than or equal to 2; and wherein at least 2 cytosine residues of the first single-stranded nucleic acid molecule are modified; and a second single-stranded nucleic acid molecule that is partially or fully complementary to the first single-stranded molecule, wherein a first label is conjugated to the first single-stranded nucleic acid molecule or the second single-stranded nucleic acid molecule; and wherein the first label is capable of producing a signal, wherein the intensity of the signal varies as a function of the conformation of the nucleic acid complex; and measuring the intensity of the signal and determining the pH from the measured signal.